Indoleacetic acid and indenacetic acid derivatives as therapeutic agents with reduced gastrointestinal toxicity

ABSTRACT

The presently disclosed subject matter provides derivatives of non-steroidal anti-inflammatory drugs (NSAIDs) that are characterized by substantially reduced cyclooxygenase inhibiting activity, yet retain the ability to interact with and modulate the activities of other polypeptides such as the class of peroxisome proliferators-activated receptors (PPARs) and γ-secretase. Also provided are methods of using the derivatives to treat pathological disorders.

CROSS REFERENCE TO RELATED APPLICATIONS

The present patent application claims benefit of U.S. patent applicationSer. No. 11/114,921, filed Apr. 26, 2005, the contents of which isincorporated herein by reference in its entirety, which itself claimsthe benefit of U.S. Provisional Application Ser. No. 60/565,489, filedApr. 26, 2004, the contents of which is also incorporated herein byreference in its entirety.

GRANT STATEMENT

This work was supported by grant number CA89450 from the U.S. NationalInstitutes of Health. Thus, the U.S. government has certain rights inthe presently disclosed subject matter.

TECHNICAL FIELD

The presently disclosed subject matter generally relates to derivativesof non-steroidal anti-inflammatory drugs (NSAIDs) that have beenmodified to decrease their ability to inhibit cyclooxygenase enzymes.Also provided are methods for altering the specificity of acyclooxygenase-inhibiting compound and methods of using the alteredcompounds to modulate various biological activities.

TABLE OF ABBREVIATIONS

-   -   15d-PGJ₂—15-deoxy-Δ^(12,14)-prostaglandin J₂    -   AA—arachidonic acid    -   AD—Alzheimer's disease    -   APP—amyloid precursor protein    -   ATCC—American Type Culture Collection    -   CCDB—Cambridge Crystallographic Data Bank    -   CNS—central nervous system    -   COX—cyclooxygenase    -   COX-1—cyclooxygenase-1    -   COX-2—cyclooxygenase-2    -   DMAP—dimethylaminopyridine    -   DMEM—Dulbecco's modified Eagle's medium    -   DM-INDO—2-Des-methylindomethacin    -   DMSO—dimethyl sulfoxide    -   DTT—dithiothreitol    -   EBA—ethyl bromoacetate    -   ED₅₀—the concentration of an compound that reduces cell        viability by 50%    -   EDCI—N-(3-dimethylaminopropyl)-N′-ethyl carbodiimide    -   EDTA—ethylenediamine tetraacetic acid    -   ESI-MS—electrospray ionization    -   Et₂O—diethyl ether    -   EtOAc—ethyl acetate    -   FBS—fetal bovine serum    -   GI—gastrointestinal    -   HOAc—acetic acid    -   IC₅₀—the concentration of an inhibitor that reduces enzyme or        cellular activity by 50%    -   INDO—indomethacin    -   mCOX-2—murine COX-2    -   MODY—maturity onset diabetes of the young    -   NSAIDs—non-steroidal anti-inflammatory drugs    -   oCOX-1—ovine COX-1    -   PG—prostaglandin    -   PMA—phosphomolybdic acid    -   PMTBA—p-methylthiobenzaldehyde    -   PPA—polyphosphoric acid    -   PPARs—peroxisome proliferator[s]-activated receptors    -   ppm—parts per million    -   PTSA—p-toluene sulfonic acid.H₂O    -   SDS—sodium dodecyl sulfate    -   SDS-PAGE—sodium dodecyl sulfate-polyacrylamide gel        electrophoresis    -   S.E.—standard error    -   TMS—tetramethylsilane

BACKGROUND

Non-steroidal anti-inflammatory drugs (NSAIDs) are a class oftherapeutic agents that are widely used for their anti-inflammatory andanti-pyretic properties to treat human distress and disease. ExemplaryNSAIDs include aspirin, ibuprofen, acetaminophen, indomethacin,naproxen, and others.

The anti-inflammatory and anti-pyretic activities of NSAIDs derive fromthe ability of these compounds to bind to and inhibit the actions of thecyclooxygenase (COX) enzymes. COX activity originates from two distinctand independently regulated enzymes, termed cyclooxygenase-1 (COX-1) andcyclooxygenase-2 (COX-2; see Dewitt & Smith, 1988; Yokoyama & Tanabe,1989; Hla & Neilson, 1992). COX-1 is a constitutive isoform and ismainly responsible for the synthesis of cytoprotective prostaglandin inthe gastrointestinal (GI) tract and for the synthesis of thromboxane,which triggers aggregation of blood platelets (Allison et al., 1992). Onthe other hand, COX-2 is inducible and short-lived. Its expression isstimulated in response to endotoxins, cytokines, and mitogens (Kujubu etal., 1991; Lee et al., 1992; O'Sullivan et al., 1993). NSAIDs exhibitvarying selectivity for COX-1 and COX-2 but, in general, most displayinhibitory activity towards both enzymes (Meade et al., 1993).

Inflammation and inflammatory responses have been associated withvarious diseases and disorders. For example, the brains of subjects withAlzheimer's disease (AD) are characterized by the accumulation ofamyloid plaques accompanied by cellular and molecular markers ofinflammatory responses. AD is the most common cause of dementia in theelderly, resulting in enormous costs to individuals and to society, bothin terms of medical care and non-economic losses. As the populationages, it is undeniable that AD and related neurological disorders willbecome an ever-increasing medical and societal burden. What is needed,then, are new and better therapeutics that can be used to prevent andtreat age-related neurological disorders.

Interestingly, epidemiological studies have suggested that long-termtreatment with NSAIDs might provide a protective effect against thedevelopment of AD. Initially, it was believed that the protective effectderived from the anti-inflammatory actions of NSAIDs, but thishypothesis has recently been questioned. Several recent reports suggestinstead that the protective effects are independent of the ability ofNSAIDs to inhibit cyclooxygenases. Thus, treatment with NSAIDs might beuseful to decrease the incidence and/or the severity of AD and relateddisorders.

Long-term use of NSAIDs is not without risks, however. In particular,most NSAIDs, particularly those that are inhibitors of COX-1, areassociated with significant GI toxicities. As such, the long-term use ofthese drugs must be approached with caution. This requires a carefulbalance between the use of NSAIDs for their potential benefits vis-à-visneurological disorders and the GI toxicity associated with their use. Amore favorable approach would be to find or create new derivatives ofNSAIDs that retain their protective effects but do not causedebilitating and potentially fatal toxicities.

One potential approach would be to employ NSAIDs that are specific forCOX-2. Several such NSAIDs have been produced, including celecoxib,valdecoxib (CELEBREX™ and BEXTRA™, respectively; Pfizer Inc., New York,N.Y., United States of America), rofecoxib, etoricoxib (VIOXX™ andARCOXIA™, respectively; Merck and Co., Inc., Whitehouse Station, N.J.,United States of America), and lumiracoxib (PREXIGE®; NovartisPharmaceuticals Corporation, East Hanover, N.J., United States ofAmerica). Unfortunately, recent evidence indicates that theseCOX-2-specific inhibitors do not provide any protective effect againstthe development of AD. In both in vivo and in vitro assays, neithercelecoxib nor rofecoxib appeared capable of inhibiting the production ofthe Aβ42 protein, the cleavage product of the amyloid precursor protein(APP) believed to be responsible for the formation of amyloid plaques.Accordingly, it appears that simply using COX-2-specific NSAIDs isunlikely to provide the protective effects currently seen with othernon-specific NSAIDs.

Additional evidence seems to suggest that the protective effectsafforded by certain NSAIDs, such as ibuprofen, sulindac sulfide, andindomethacin (all non-specific NSAIDs), might not be related to theirCOX-inhibition activities, and thus might be related to the abilities ofthese NSAIDs to interact with other polypeptides present in the centralnervous system (CNS). Two such polypeptides are the class of peroxisomeproliferators-activated receptors (PPARs) and γ-secretase. For example,PPARs, particularly PPARγ, have been implicated in mediatingdifferentiation of adipocytes and regulating fat metabolism.Additionally, PPARγ has been associated with various pathologicalconditions related to atherosclerosis, inflammation, obesity, diabetes,cancer, the immune response, and ageing. See Kersten et al., 2000; Celi& Shuldiner, 2002. γ-secretase, on the other hand, appears to be themain enzyme responsible for the production of Aβ42 from APP, and thushas a critical role in the pathogenesis of AD.

What are needed, then, are new derivatives of NSAIDs that are less toxicthan the parent NSAIDs, yet retain the abilities of the parents tomodulate the activities of, for example, PPARs and/or γ-secretase. Thisand other needs are addressed by the compositions and methods of thepresently disclosed subject matter.

SUMMARY

This Summary lists several embodiments of the presently disclosedsubject matter, and in many cases lists variations and permutations ofthese embodiments. This Summary is merely exemplary of the numerous andvaried embodiments. Mention of one or more representative features of agiven embodiment is likewise exemplary. Such an embodiment can typicallyexist with or without the feature(s) mentioned; likewise, those featurescan be applied to other embodiments of the presently disclosed subjectmatter, whether listed in this Summary or not. To avoid excessiverepetition, this Summary does not list or suggest all possiblecombinations of such features.

The presently disclosed subject matter provides a method for inhibitinggrowth of a cell. In some embodiments, the method comprises contactingthe cell with a derivative of a compound, wherein the compound comprisesa cyclooxygenase inhibitor comprising an indoleacetic acid orindenacetic acid functional group having a 2′ methyl group and thederivative substantially lacks cyclooxygenase inhibitory activity as aresult of modifying the 2′ methyl group to a moiety selected from thegroup consisting of hydrogen; halo; halomethyl, wherein at least onehydrogen of the methyl group is substituted with a halogen; C₂ to C₆alkyl; C₂ to C₆ branched alkyl; and C₂ to C₆ substituted alkyl. In someembodiments, the cyclooxygenase inhibitor comprises an indenacetic acidfunctional group and the moiety is selected from the group consisting ofhydrogen and fluorine. In some embodiments, the cell is present in asubject. In some embodiments, the cell is a tumor cell. In someembodiments, the subject is a mammal. In some embodiments, the mammal isa human.

In some embodiments, the compound is a non-steroidal anti-inflammatorydrug. In some embodiments, the non-steroidal anti-inflammatory drug isselected from the group consisting of indomethacin, sulindac,pharmaceutically acceptable salts thereof, and combinations thereof. Insome embodiments, the derivative of the compound is an amide or esterderivative.

The presently disclosed subject matter also provides a method fortreating a disease in a subject selected from the group consisting of acancer, a neurodegenerative disease, and diabetes. In some embodiments,the method comprises administering to the subject a treatment effectiveamount of a derivative of a compound, wherein the compound comprises acyclooxygenase inhibitor comprising an indoleacetic acid or indenaceticacid functional group having a 2′ methyl group and the derivativesubstantially lacks cyclooxygenase inhibitory activity as a result ofmodifying the 2′ methyl group to a moiety selected from the groupconsisting of hydrogen; halo; halomethyl, wherein at least one hydrogenof the methyl group is substituted with a halogen; C₂ to C₆ alkyl; C₂ toC₆ branched alkyl; and C₂ to C₆ substituted alkyl. In some embodiments,the cyclooxygenase inhibitor comprises an indenacetic acid functionalgroup and the moiety is selected from the group consisting of hydrogenand fluorine. In some embodiments, the subject is a mammal. In someembodiments, the mammal is a human. In some embodiments, theneurodegenerative disease is Alzheimer's disease.

In some embodiments, the compound is a non-steroidal anti-inflammatorydrug. In some embodiments, the non-steroidal anti-inflammatory drug isselected from the group consisting of indomethacin and sulindac, andpharmaceutically acceptable salts thereof, and combinations thereof. Insome embodiments, the derivative of the compound is an amide or esterderivative. In some embodiments, the derivative causes substantiallyless gastrointestinal toxicity than does the compound. In someembodiments, the derivative of the compound is an amide or esterderivative.

The presently disclosed subject matter also provides a method forsuppressing tumor growth in a subject. In some embodiments, the methodcomprises administering to a subject bearing a tumor a derivative of acompound, wherein the compound comprises a cyclooxygenase inhibitorcomprising an indoleacetic acid or indenacetic acid functional grouphaving a 2′ methyl group and the derivative substantially lackscyclooxygenase inhibitory activity as a result of modifying the 2′methyl group to a moiety selected from the group consisting of hydrogen;halo; halomethyl, wherein at least one hydrogen of the methyl group issubstituted with a halogen; C₂ to C₆ alkyl; C₂ to C₆ branched alkyl; andC₂ to C₆ substituted alkyl. In some embodiments, the cyclooxygenaseinhibitor comprises an indenacetic acid functional group and the moietyis selected from the group consisting of hydrogen and fluorine. In someembodiments, the subject is a mammal. In some embodiments, the mammal isa human.

In some embodiments, the compound is a non-steroidal anti-inflammatorydrug. In some embodiments, the non-steroidal anti-inflammatory drug isselected from the group consisting of indomethacin and sulindac, andpharmaceutically acceptable salts thereof, and combinations thereof. Insome embodiments, the derivative of the compound is an amide or esterderivative. In some embodiments, the derivative causes substantiallyless gastrointestinal toxicity than does the compound.

The presently disclosed subject matter also provides a method forinducing apoptosis in a cell. In some embodiments, the method comprisescontacting the cell with a derivative of a compound, wherein thecompound comprises a cyclooxygenase inhibitor comprising an indoleaceticacid or indenacetic acid functional group having a 2′ methyl group andthe derivative substantially lacks cyclooxygenase inhibitory activity asa result of modifying the 2′ methyl group to a moiety selected from thegroup consisting of hydrogen; halo; halomethyl, wherein at least onehydrogen of the methyl group is substituted with a halogen; C₂ to C₆alkyl; C₂ to C₆ branched alkyl; and C₂ to C₆ substituted alkyl. In someembodiments, the cyclooxygenase inhibitor comprises an indenacetic acidfunctional group and the moiety is selected from the group consisting ofhydrogen and fluorine. In some embodiments, the cell is a cell inculture. In some embodiments, the cell is a cancer cell. In someembodiments, the cell is present within a subject. In some embodiments,the subject is a mammal. In some embodiments, the mammal is a human.

In some embodiments, the compound is a non-steroidal anti-inflammatorydrug. In some embodiments, the non-steroidal anti-inflammatory drug isselected from the group consisting of indomethacin and sulindac, andpharmaceutically acceptable salts thereof, and combinations thereof. Insome embodiments, the derivative of the compound is an amide or esterderivative.

The presently disclosed subject matter also provides a method formodulating the activity of a peroxisome proliferators-activated receptor(PPAR) isoform. In some embodiments, the method comprises contacting thePPAR isoform with a derivative of a compound, wherein the compoundcomprises a cyclooxygenase inhibitor comprising an indoleacetic acid orindenacetic acid functional group having a 2′ methyl group and thederivative substantially lacks cyclooxygenase inhibitory activity as aresult of modifying the 2′ methyl group to a moiety selected from thegroup consisting of hydrogen; halo; halomethyl, wherein at least onehydrogen of the methyl group is substituted with a halogen; C₂ to C₆alkyl; C₂ to C₆ branched alkyl; and C₂ to C₆ substituted alkyl. In someembodiments, the cyclooxygenase inhibitor comprises an indenacetic acidfunctional group and the moiety is selected from the group consisting ofhydrogen and fluorine. In some embodiments, the PPAR isoform is PPARγ.In some embodiments, the PPAR isoform is present within a subject. Insome embodiments, the subject is a mammal. In some embodiments, themammal is a human.

In some embodiments, the compound is a non-steroidal anti-inflammatorydrug. In some embodiments, the non-steroidal anti-inflammatory drug isselected from the group consisting of indomethacin, sulindac,pharmaceutically acceptable salts thereof, and combinations thereof. Insome embodiments, the derivative of the compound is an amide or esterderivative. In some embodiments, the derivative causes substantiallyless gastrointestinal toxicity than does the compound.

The presently disclosed subject matter also provides a method foraltering specificity of a cyclooxygenase-inhibiting compound. In someembodiments, the method comprises (a) providing a compound havingcyclooxygenase inhibitory activity, the compound comprising anindoleacetic acid or indenacetic acid functional group having a 2′methyl group; and (b) replacing the 2′ methyl group with a moietyselected from the group consisting of hydrogen; halo; halomethyl,wherein at least one hydrogen of the methyl group is substituted with ahalogen; C₂ to C₆ alkyl; C₂ to C₆ branched alkyl; and C₂ to C₆substituted alkyl to create a derivative, wherein the derivativesubstantially lacks cyclooxygenase inhibitory activity. In someembodiments, the compound is a non-steroidal anti-inflammatory drug. Insome embodiments, the non-steroidal anti-inflammatory drug is selectedfrom the group consisting of indomethacin and sulindac, andpharmaceutically acceptable salts thereof, and combinations thereof. Insome embodiments, the derivative is selected from the group consistingof 2-Des-methylindomethacin and eindenic acid sulfide, eindenic acidsulfoxide, and eindenic acid sulfone. In some embodiments, thederivative is eindenic acid sulfide. In some embodiments, the presentmethod further comprises derivatizing a carboxylic acid moiety presenton the compound to an ester or an amide.

The presently disclosed subject matter also provides compositions thatcan be used in conjunction with any or all of the disclosed methods. Insome embodiments, the presently disclosed subject matter provides acompound of the following formula:

wherein

-   -   R¹ is selected from the group consisting of hydrogen, halo, CF₃;        SCH₃; SOCH₃; SO₂CH₃; SO₂NH₂; C₁ to C₆ alkyl, branched alkyl, or        substituted alkyl; C₁ to C₆ alkoxy, branched alkoxy, or        substituted alkoxy; C₁ to C₆ alkylcarboxylic acid, branched        alkylcarboxylic acid, or substituted alkylcarboxylic acid; and        CH₂N₃;    -   R² is selected from the group consisting of hydrogen, halo, CF₃;        SCH₃; SOCH₃; SO₂CH₃; SO₂NH₂; CONH₂; C₁ to C₆ alkyl, branched        alkyl, or substituted alkyl; C₁ to C₆ alkoxy, branched alkoxy,        or substituted alkoxy; benzyloxy; C₁ to C₆ alkylcarboxylic acid,        branched alkylcarboxylic acid, or substituted alkylcarboxylic        acid; and CH₂N₃;    -   R³ and R⁴ are each independently selected from the group        consisting of hydrogen; halo; CF₃; C₁ to C₆ alkyl, branched        alkyl, or substituted alkyl; C₁ to C₆ alkoxy, branched alkoxy,        or substituted alkoxy; aryl; substituted aryl; benzyloxy; SCH₃;        SOCH₃; SO₂CH₃; and SO₂NH₂;    -   R⁵ is selected from the group consisting of hydrogen, C₁ to C₆        alkyl, branched alkyl, or substituted alkyl, and ═O;    -   R⁶ is selected from the group consisting of hydrogen; C₁ to C₆        alkyl, branched alkyl, or substituted alkyl; C₁ to C₆ alkoxy,        branched alkoxy, or substituted alkoxy; benzyloxy; C₁ to C₆        alkylcarboxylic acid, branched alkylcarboxylic acid, or        substituted alkylcarboxylic acid; and the following structure:

-   -   wherein        -   Ar is cyclohexyl or phenyl;        -   R⁷ is hydrogen; C₁ to C₆ alkyl, branched alkyl, or            substituted alkyl;        -   R⁸ is hydrogen, halo, C₁ to C₆ alkyl, branched alkyl, and            substituted alkyl; C₁ to C₆ alkoxy, branched alkoxy, and            substituted alkoxy; C₁ to C₆ alkylcarboxylic acid, branched            alkylcarboxylic acid, or substituted alkylcarboxylic acid;            amino; nitro; CF₃; bromoacetamidyl; benzoyl; or            2-phenyl-oxiranyl;        -   X is O or NR⁹, wherein R⁹ is hydrogen or alkyl; and        -   m, n, s, and t are each individually 0, 1, 2, 3, 4, or 5;    -   Y is selected from the group consisting of hydrogen, halo, CF₃,        and C₂ to C₆ alkyl or branched alkyl;    -   A is selected from the group consisting of carbon and nitrogen;    -   p and q are both individually 0, 1, 2, 3, or 4;    -   the bond between the carbon bound to R⁵ and the indene ring and        is a single bond or a double bond; and    -   the six-membered ring to which R¹ is bound is cyclohexyl or        phenyl.

In some embodiments, R¹ is selected from the group consisting of halo,C₁ to C₆ alkyl or branched alkyl, SCH₃, SOCH₃, SO₂CH₃, and SO₂NH₂; R² isselected from the group consisting of hydrogen; halo; C₁ to C₆ alkyl orbranched alkyl; C₁ to C₆ alkoxy or branched alkoxy; benzyloxy; SCH₃;SOCH₃; SO₂CH₃; SO₂NH₂; and CONH₂; R³ and R⁴ are each independentlyselected from the group consisting of hydrogen, C₁ to C₆ alkyl orbranched alkyl, and halo; R⁵ is selected from the group consisting ofhydrogen, C₁ to C₆ alkyl or branched alkyl, and carbonyl; R⁶ is selectedfrom the group consisting of C₁ to C₆ alkylcarboxylic acid and branchedC₁ to C₆ alkylcarboxylic acid; Y is selected from the group consistingof hydrogen, halo, and C₂ to C₆ alkyl or branched alkyl; A is selectedfrom the group consisting of carbon and nitrogen; and the bond betweenthe carbon bound to R⁵ and the indene ring is a single bond or a doublebond. In some embodiments, the derivative is selected from the groupconsisting of 2-Des-methylindomethacin and eindenic acid sulfide,eindenic acid sulfoxide, and eindenic acid sulfone. In some embodiments,the derivative is eindenic acid sulfide.

In some embodiments, the compound has the following formula:

In some embodiments, the compound has the following formula:

In some embodiments, the compound has the following formula:

In some embodiments, the presently disclosed subject matter provides acompound of one of the following formulas:

wherein

-   -   R² is selected from the group consisting of hydrogen, halo, CF₃;        SCH₃; SOCH₃; SO₂CH₃; SO₂NH₂; CONH₂; C₁ to C₆ alkyl, branched        alkyl, or substituted alkyl; C₁ to C₆ alkoxy, branched alkoxy,        or substituted alkoxy; benzyloxy; C₁ to C₆ alkylcarboxylic acid,        branched alkylcarboxylic acid, or substituted alkylcarboxylic        acid; and CH₂N₃;    -   R⁴ is selected from the group consisting of hydrogen; halo; CF₃;        C₁ to C₆ alkyl, branched alkyl, or substituted alkyl; C₁ to C₆        alkoxy, branched alkoxy, or substituted alkoxy; aryl;        substituted aryl; benzyloxy; SCH₃; SOCH₃; SO₂CH₃; and SO₂NH₂;    -   R⁵ is selected from the group consisting of hydrogen, C₁ to C₆        alkyl, C₁ to C₆ branched alkyl, and C₁ to C₆ substituted alkyl,        and in the case of Formula Ia, ═O;    -   R⁶ is selected from the group consisting of hydrogen; C₁ to C₆        alkyl, branched alkyl, or substituted alkyl; C₁ to C₆ alkoxy,        branched alkoxy, or substituted alkoxy; benzyloxy; C₁ to C₆        alkylcarboxylic acid, branched alkylcarboxylic acid, or        substituted alkylcarboxylic acid; and the following structure:

-   -   wherein        -   Ar is cyclohexyl or phenyl;        -   R⁷ is hydrogen; C₁ to C₆ alkyl, branched alkyl, or            substituted alkyl;        -   R⁸ is hydrogen, halo, C₁ to C₆ alkyl, branched alkyl, and            substituted alkyl; C₁ to C₆ alkoxy, branched alkoxy, and            substituted alkoxy; C₁ to C₆ alkylcarboxylic acid, branched            alkylcarboxylic acid, or substituted alkylcarboxylic acid;            amino; nitro; CF₃; bromoacetamidyl; benzoyl; or            2-phenyl-oxiranyl;        -   X is O or NR⁹, wherein R⁹ is hydrogen or alkyl; and        -   m, n, and t are each individually 0, 1, 2, 3, 4, or 5;    -   R¹⁵ is selected from the group consisting of cyclohexyl and an        aromatic substituent, wherein the aromatic substituent comprises        an aryl, a heteroaryl, and singly or multiply substituted        derivatives thereof;    -   Y is selected from the group consisting of hydrogen; halo;        halomethyl, wherein at least one hydrogen of the methyl group is        substituted with a halogen; C₂ to C₆ alkyl; C₂ to C₆ branched        alkyl; and C₂ to C₆ substituted alkyl; and    -   q is 0, 1, 2, 3, or 4.

In some embodiments, R¹⁵ comprises an aryl comprising multiple aromaticrings that are fused together or covalently linked. In some embodiments,the multiple aromatic rings are covalently linked by a moiety selectedfrom the group consisting of an alkylene moiety, a carbonyl, oxygen,diphenylether, and nitrogen. In some embodiments, the multiple aromaticrings are selected from the group consisting of naphthyl, biphenyl,diphenylether, diphenylamine, and benzophenone.

In some embodiments, R² is selected from the group consisting of halo,C₁ to C₆ alkyl or branched alkyl, SCH₃, SOCH₃, SO₂CH₃, and SO₂NH₂. Insome embodiments, R² is F.

In some embodiments, R¹⁵ is in the E orientation with respect to theindene ring. In some embodiments, R³ comprises one of the followingformulas:

In some embodiments, the compound has one of the following formulas:

In some embodiments, the method further comprises derivatizing acarboxylic acid moiety present on the compound to an amide. In someembodiments, the amide derivative has the following general formula:

In some embodiments, R¹¹ is selected from the group consisting of C₁ toC₆ alkyl, branched alkyl, and cyclic alkyl. In some embodiments, R¹¹ isselected from the group consisting of C₁ to C₆ alkylcarboxylic acid,branched alkylcarboxylic acid, and cyclic alkylcarboxylic acid. In someembodiments, R¹¹ is selected from the group consisting of C₁ to C₆ aryland C₁ to C₆ substituted aryl. In some embodiments of the substitutedaryl, the substitution is at least one position, and each substitutionis selected from the group consisting of a halogen, NH₂, OCH₃, CF₃, OH,C₁ to C₄ alkyl or branched alkyl, NO₂, benzoyl, 2-phenyl-oxirane, andNH—CO—CH₂Br.

In some embodiments, the amide derivative has the following generalformula:

wherein R¹² is selected from the group consisting of phenyl-SOCH₃,phenyl-SO₂CH₃, phenyl, phenyl methyl ester, phenyl-COOH, phenyl-halo,and C₃ to C₆ cycloalkyl. Representative amide derivatives are presentedin Tables 1 and 2.

In some embodiments, the method further comprises derivatizing acarboxylic acid moiety present on the compound to an ester or an amide.In some embodiments, the ester or amide has the following generalformula:

wherein R² is defined as above, R¹³ is selected from the groupconsisting of C₁ to C₆ alkyl, C₁ to C₆ branched alkyl, and C₁ to C₆substituted alkyl. In some embodiments, the ester derivative has thefollowing formula:

Accordingly, it is an object of the presently disclosed subject matterto provide new therapeutic agents for use in treating and/or preventingdisease. This object is achieved in whole or in part by the presentlydisclosed subject matter.

An object of the presently disclosed subject matter having been statedhereinabove, other objects will be evident as the description proceedsand as best described hereinbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent with color drawing(s) will be provided bythe Patent and Trademark Office upon request and payment of necessaryfee.

FIGS. 1A-1C depict a crystal structure of indomethacin (INDO) bound inthe COX-2 active site.

FIGS. 1A and 1B depict stereo views of INDO co-crystallized with COX-2(Protein Data Bank code 4COX; Kurumbail et al., 1996). In FIG. 1A, keyactive site residues for catalysis and the binding of ligands are shown.FIG. 1B is a space-filling model of the 2′ methyl substituent of INDO(green) inserted into the hydrophobic pocket formed by Val-349, Ala-527,Ser-530, and Leu-531.

FIG. 1C depicts the chemical structures of INDO and DM-INDO.

FIGS. 2A-2D depict the kinetics of the time-dependent inhibition ofCOX-2 mutants by INDO. Assays were performed with various concentrationsof either INDO or DM-INDO as described in Example 7.

FIGS. 2A and 2C depict representative data expressed as percent activityof the uninhibited control with non-linear regression curves. The curvesdrawn as secondary plots for FIG. 2B and FIG. 2D were generated byfitting the data presented in FIGS. 2A and 2C, respectively, to equation(2), disclosed hereinbelow.

FIGS. 3A and 3B depict the effects of the three Val-349 mutations on thereversibility of COX-2 inhibition by INDO and DM-INDO. Assays wereperformed with 10 μM of either INDO or DM-INDO as described in Example7. Representative data are expressed as percent activity of theuninhibited control.

FIGS. 4A and 4B depict the kinetics of the time-dependent inhibition ofCOX-2 mutants by DM-INDO. Assays were performed as described in Example7. Representative data are expressed as percent activity of theuninhibited control with non-linear regression curves.

FIGS. 5A-5D depict fluorescence quenching of apo-COX-2 by INDO comparedto DM-INDO, and competition by arachidonic acid (AMINO ACID). Assayswere performed under conditions described in Example 9. Apo-protein at0.2 μM was mixed with DMSO (black), INDO (red), or DM-INDO (blue) for240 seconds (FIGS. 5A-5C) or 360 seconds (FIG. 5D), followed by theaddition of 50 μM AA (arrow), and monitored for another 240 seconds(FIGS. 5A-5C) or 360 seconds (FIG. 5D). Traces are the average of 3determinations.

FIG. 5A depicts the results obtained with an mCOX-2^(V349A) polypeptide.FIG. 5B depicts the results obtained with a wild type mCOX-2polypeptide. FIG. 5C depicts the results obtained with an mCOX-2^(V349I)polypeptide. FIG. 5D depicts the results obtained with an mCOX-2^(V349L)polypeptide. The final concentrations of INDO and DM-INDO employed were1 μM (FIG. 5A), 2 μM (FIG. 5B),) 3 μM (FIG. 5C), and 5 μM (FIG. 5D).

FIG. 6 depicts a scheme for synthesizing 2-Des-methylindomethacin(DM-INDO).

FIG. 7 depicts a scheme for synthesizing eindenic acid sulfide (CompoundI) and a derivative of eindenic acid sulfide,N-Benzyl-2-[6-fluoro-3-(4-methylsulfanyl-benzylidene)-3H-inden-1-yl]-acetamide(Compound J).

FIG. 8 depicts the results of cell viability assays of RKO cells exposedto various concentrations ofN-Benzyl-2-[6-fluoro-3-(4-methylsulfanyl-benzylidene)-3H-inden-1-yl]-acetamide(Compound J).

FIG. 9 depicts the results of increased caspase-3 activity in threedifferent cell lines exposed to various concentrations ofN-Benzyl-2-[6-fluoro-3-(4-methylsulfanyl-benzylidene)-3H-inden-1-yl]-acetamide(Compound J).

FIG. 10 depicts the Western blot analyses of results of PPARγ reporterassays of HEK293 cells exposed to various concentrations of sulindacsulfide (SS), eindenic acid sulfide (Compound I), orN-Benzyl-2-[6-fluoro-3-(4-methylsulfanyl-benzylidene)-3H-inden-1-yl]-acetamide(Compound J).

DETAILED DESCRIPTION

The present subject matter will be now be described more fullyhereinafter with reference to the accompanying Examples, in whichrepresentative embodiments of the presently disclosed subject matter areshown. The presently disclosed subject matter can, however, be embodiedin different forms and should not be construed as limited to theembodiments set forth herein. Rather, these embodiments are provided sothat this disclosure will be thorough and complete, and will fullyconvey the scope of the presently disclosed subject matter to thoseskilled in the art.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety.

Throughout the specification and claims, a given chemical formula orname shall encompass all optical isomers and stereoisomers as well asracemic mixtures where such isomers and mixtures exist.

I. GENERAL CONSIDERATIONS

Non-steroidal anti-inflammatory drugs (NSAIDs) exert a range ofbiological activities including inhibition of inflammation, inhibitionof pain, lowering of fever, inhibition of tumor growth, inhibition ofAlzheimer's disease, and improvement of cognitive function inneurodegenerative diseases, inter alia. Some of these effects aremediated by inhibition of cyclooxygenase enzymes (COX-1 and COX-2)whereas others are mediated by modulation of other molecular targets.The latter include, but are not limited to activation of peroxisomeproliferators-activated receptors (PPARs), modulation of γ-secretase,inhibition of c-GMP phosphodiesterase subtypes, and inhibition of Rhoactivation. Two compounds that exhibit activities in bothcyclooxygenase-related and non-cyclooxygenase-related responses areindomethacin and sulindac sulfide. Indomethacin is directly activefollowing administration to humans whereas sulindac sulfide isadministered as the inactive prodrug sulindac. Sulindac is converted tothe active drug, sulindac sulfide, by reduction in the gastrointestinaltract.

Indomethacin and sulindac sulfide are structurally related moleculesthat contain substituted indoleacetic acid and indeneacetic acidfunctional groups, respectively. Both molecules contain a methyl groupat the 2-position of the indole or indene ring. Molecular modelingsuggests that this 2′ methyl group is an important determinant of theability of indomethacin and sulindac sulfide to bind tightly to COXenzymes and, thereby, inhibit their function. This hypothesis has beenverified by site-directed mutagenesis of the COX-2 enzyme and bysynthesis of indomethacin and sulindac sulfide derivatives that lack the2′ methyl group (2-Des-methyl derivatives) as described herein. Thesederivatives are poor inhibitors of both COX enzymes compared to theparent drugs.

The inability of 2-Des-methyl derivatives of indole acetic acids andindene acetic acids to inhibit COX enzymes provides a strategy todevelop drugs that display COX-independent effects but minimally inhibitCOX enzymes and, therefore, have a higher safety margin by virtue ofreduced gastrointestinal toxicity. This enables higher doses of thesedrugs to be given, which should increase their efficacy at non-COXtargets. These compounds would be expected to exhibit the ability toprevent, treat, or inhibit cancer, neurodegenerative diseases, anddiabetes inter alia.

II. DEFINITIONS

While the following terms are believed to be well understood by one ofordinary skill in the art, the following definitions are set forth tofacilitate explanation of the presently disclosed subject matter.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which the presently disclosed subject matter belongs.Although any methods, devices, and materials similar or equivalent tothose described herein can be used in the practice or testing of thepresently disclosed subject matter, representative methods, devices, andmaterials are now described.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about”. Accordingly, unless indicated to the contrary, thenumerical parameters set forth in this specification and attached claimsare approximations that can vary depending upon the desired propertiessought to be obtained by the presently disclosed subject matter.

Following long-standing patent law convention, the terms “a”, “an”, and“the” refer to “one or more” when used in this application, includingthe claims. Thus, for example, reference to “a vector” includes aplurality of such vectors, and so forth.

As used herein, the term “about,” when referring to a value or to anamount of mass, weight, time, volume, concentration or percentage ismeant to encompass variations of in some embodiments ±20%, in someembodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, insome embodiments ±0.5%, and in some embodiments ±0.1% from the specifiedamount, as such variations are appropriate to perform the disclosedmethod.

As used herein, the terms “amino acid” and “amino acid residue” are usedinterchangeably and mean any of the twenty naturally occurring aminoacids. An amino acid is formed upon chemical digestion (hydrolysis) of apolypeptide at its peptide linkages. The amino acid residues describedherein are in some embodiments in the “L” isomeric form. However,residues in the “D” isomeric form can be substituted for any L-aminoacid residue, as long as the desired functional property is retained bythe polypeptide. NH₂ refers to the free amino group present at the aminoterminus of a polypeptide. COOH refers to the free carboxy group presentat the carboxy terminus of a polypeptide. In keeping with standardpolypeptide nomenclature abbreviations for amino acid residues are shownin tabular form presented hereinabove.

It is noted that all amino acid residue sequences represented herein byformulae have a left-to-right orientation in the conventional directionof amino terminus to carboxy terminus. In addition, the phrases “aminoacid” and “amino acid residue” are broadly defined to include modifiedand unusual amino acids.

Furthermore, it is noted that a dash at the beginning or end of an aminoacid residue sequence indicates a peptide bond to a further sequence ofone or more amino acid residues or a covalent bond to an amino-terminalgroup such as NH₂ or acetyl or to a carboxy-terminal group such as COOH.

In certain instances herein, amino acids are indicated by a one- orthree-letter code followed by a number (for example, Val-349). As usedherein, this numbering system refer to the positions of correspondingamino acids in ovine COX-1, the amino acid sequence of which can befound at GENBANK® Accession No. P05979. As a result of this convention,a given amino acid and number combination might not be found in a givenpolypeptide, depending on the particular COX enzyme and species. Forexample, “Val-349” refers to the valine residue that forms part of thebinding pocket for the 2′-methyl group of indomethacin or sulindac.Looking at GENBANK® Accession No. P05979, one can find a valine atposition 349. However, when looking at GENBANK® Accession No. Q05769,which is the mouse COX-2 amino acid sequence, one finds that thecorresponding valine is not at amino acid 349, but rather at amino acid335. Similarly, Ala-527, Ser-530, and Leu-531 refer not only to aminoacids at positions 527, 530, and 531 of ovine COX-1, respectively, butalso to alanine, serine, and leucine residues found in mouse COX-2 atpositions 513, 516, and 517, respectively. The human COX-2 amino acidsequence can be found at GENBANK® Accession No. P35354, and in humanCOX-2, Val-349, Ala-527, Ser-530, and Leu-531 also refer to the valine,alanine, serine, and leucine amino acids that are present at amino acids335, 513, 516, and 517, respectively.

As used herein, the term “cell” refers not only to the particularsubject cell (e.g., a living biological cell), but also to the progenyor potential progeny of such a cell. Because certain modifications canoccur in succeeding generations due to either mutation or environmentalinfluences, such progeny might not, in fact, be identical to the parentcell, but are still included within the scope of the term as usedherein.

As used herein, the term “enzyme activity” refers to the ability of anenzyme to catalyze the conversion of a substrate into a product. Asubstrate for the enzyme can comprise the natural substrate of theenzyme but also can comprise analogues of the natural substrate, whichcan also be converted by the enzyme into a product or into an analogueof a product. The activity of the enzyme is measured for example bydetermining the amount of product in the reaction after a certain periodof time, or by determining the amount of substrate remaining in thereaction mixture after a certain period of time. The activity of theenzyme can also be measured by determining the amount of an unusedco-factor of the reaction remaining in the reaction mixture after acertain period of time or by determining the amount of used co-factor inthe reaction mixture after a certain period of time. The activity of theenzyme can also be measured by determining the amount of a donor of freeenergy or energy-rich molecule (e.g., ATP, phosphoenolpyruvate, acetylphosphate, or phosphocreatine) remaining in the reaction mixture after acertain period of time or by determining the amount of a used donor offree energy or energy-rich molecule (e.g., ADP, pyruvate, acetate, orcreatine) in the reaction mixture after a certain period of time.

As used herein, the term “inhibitor” refers to a chemical substance thatinactivates or decreases the biological activity of a polypeptide suchas a biosynthetic and catalytic activity, receptor, signal transductionpolypeptide, structural gene product, or transport polypeptide.

As used herein, the term “interact” includes “binding” interactions and“associations” between molecules. Interactions can be, for example,protein-protein, protein-small molecule, protein-nucleic acid, andnucleic acid-nucleic acid in nature.

As used herein, the term “modulate” refers to an increase, decrease, orother alteration of any, or all, chemical and biological activities orproperties of a biochemical entity, e.g., a wild type or mutantpolypeptide. As such, the term “modulate” can refer to a change in theexpression level of a gene (or a level of RNA molecule or equivalent RNAmolecules encoding one or more proteins or protein subunits), or of anactivity of one or more proteins or protein subunits, such thatexpression, level, or activity is greater than or less than thatobserved in the absence of the modulator. For example, the term“modulate” can mean “inhibit” or “suppress”, but the use of the word“modulate” is not limited to this definition.

The term “modulation” as used herein refers to both upregulation (i.e.,activation or stimulation) and downregulation (i.e., inhibition orsuppression) of a response. Thus, the term “modulation”, when used inreference to a functional property or biological activity or process(e.g., enzyme activity or receptor binding), refers to the capacity toupregulate (e.g., activate or stimulate), downregulate (e.g., inhibit orsuppress), or otherwise change a quality of such property, activity, orprocess. In certain instances, such regulation can be contingent on theoccurrence of a specific event, such as activation of a signaltransduction pathway, and/or can be manifest only in particular celltypes.

The term “modulator” refers to a polypeptide, nucleic acid,macromolecule, complex, molecule, small molecule, compound, species, orthe like (naturally occurring or non-naturally occurring) that can becapable of causing modulation. Modulators can be evaluated for potentialactivity as inhibitors or activators (directly or indirectly) of afunctional property, biological activity or process, or a combinationthereof, (e.g., agonist, partial antagonist, partial agonist, inverseagonist, antagonist, anti-microbial agents, inhibitors of microbialinfection or proliferation, and the like) by inclusion in assays. Insuch assays, many modulators can be screened at one time. The activityof a modulator can be known, unknown, or partially known.

Modulators can be either selective or non-selective. As used herein, theterm “selective” when used in the context of a modulator (e.g., aninhibitor) refers to a measurable or otherwise biologically relevantdifference in the way the modulator interacts with one molecule (e.g.,an enzyme or receptor) versus another similar but not identical molecule(e.g., a member of the same enzyme or receptor family).

It must be understood that it is not required that the degree to whichthe interactions differ be completely opposite. Put another way, theterm selective modulator encompasses not only those molecules that onlybind to a given polypeptide and not to related family members. The termis also intended to include modulators that are characterized byinteractions with polypeptides of interest and from related familymembers that differ to a lesser degree. For example, selectivemodulators include modulators for which conditions can be found (such asthe nature of the substituents present on the modulator) that wouldallow a biologically relevant difference in the binding of the modulatorto the polypeptide of interest versus polypeptides derived fromdifferent family members.

When a selective modulator is identified, the modulator will bind to onemolecule (for example a polypeptide of interest) in a manner that isdifferent (for example, stronger) than it binds to another molecule (forexample, a polypeptide related to the polypeptide of interest). As usedherein, the modulator is said to display “selective binding” or“preferential binding” to the molecule to which it binds more strongly.

As used herein, the term “mutation” carries its traditional connotationand means a change, inherited, naturally occurring or introduced, in anucleic acid or polypeptide sequence, and is used in its sense asgenerally known to those of skill in the art.

As used herein, the terms “nucleic acid” and “nucleic acid molecule”mean any of deoxyribonucleic acid (DNA), ribonucleic acid (RNA),oligonucleotides, fragments generated by the polymerase chain reaction(PCR), and fragments generated by any of ligation, scission,endonuclease action, and exonuclease action. Nucleic acids can becomposed of monomers that are naturally occurring nucleotides (such asdeoxyribonucleotides and ribonucleotides), or analogs of naturallyoccurring nucleotides (e.g., α-enantiomeric forms of naturally-occurringnucleotides), or a combination of both. Nucleic acids can be eithersingle stranded or double stranded.

As used herein, the term “polypeptide” means any polymer comprising anyof the 20 protein amino acids, or amino acid analogs, regardless of itssize or function. Although “protein” is often used in reference torelatively large polypeptides, and “peptide” is often used in referenceto small polypeptides, usage of these terms in the art overlaps andvaries. The term “polypeptide” as used herein refers to peptides,polypeptides and proteins, unless otherwise noted. As used herein, theterms “protein”, “polypeptide” and “peptide” are used interchangeably.The term “polypeptide” encompasses proteins of all functions, includingenzymes.

As used herein, the terms “polypeptides of interest” and “targetpolypeptide” are used interchangeably to refer to a polypeptide theactivity of which the compositions and methods of the presentlydisclosed subject matter are intended to modulate. For example,polypeptides of interest include, but are not limited to cyclooxygenaseenzymes, PPARs (e.g., PPARγ), and secretases (e.g., γ-secretase). Thecompositions disclosed herein are intended to differentially modulatethese enzymes relative to one or more of the others. For example, theNSAID derivatives disclosed herein are intended to have reducedcyclooxygenase-binding activities, while their binding activities toother polypeptides might or might not be affected by the derivitization.While not wishing to be limited to any particular theory of operation,the reduction in COX-binding activity might enhance the bioavailabilityof these derivatives to other, non-COX polypeptides of interest becausethe derivatives either do not bind to COX enzymes or bind to COX enzymesto a lesser degree than do the non-derivatized NSAIDs upon which theyare based.

As used herein, “significance” or “significant” relates to a statisticalanalysis of the probability that there is a non-random associationbetween two or more entities. To determine whether or not a relationshipis “significant” or has “significance”, statistical manipulations of thedata can be performed to calculate a probability, expressed as a“p-value”. Those p-values that fall below a user-defined cutoff pointare regarded as significant. In one example, a p-value less than orequal to 0.05, in another example less than 0.01, in another exampleless than 0.005, and in yet another example less than 0.001, areregarded as significant.

As used herein, the term “significant increase” refers to an increase inactivity (for example, enzymatic activity) that is larger than themargin of error inherent in the measurement technique, in someembodiments an increase by about 2 fold or greater over a baselineactivity (for example, the activity of the wild type enzyme in thepresence of the inhibitor), in some embodiments an increase by about 5fold or greater, and in still some embodiments an increase by about 10fold or greater.

With respect to the binding of one or more molecules (for example, amodulator) to one or more polypeptides (for example, a PPAR, a COX, or asecretase), a significant increase can also refer to: (a) a biologicallyrelevant difference in binding of two or more related compounds to thesame polypeptide; and/or (b) a biologically relevant difference inbinding of the same compound to two different polypeptides. In thisaspect, “significant” is to be thought of in its ordinary meaning:namely, a difference between two occurrences that is important (i.e.,biologically or medically relevant). By way of example, a significantincrease can also refer to an increase in the amount of a derivative ofan NSAID (for example, a 2-Des-methyl derivative of the presentlydisclosed subject matter) that interacts with a non-COX polypeptide (forexample, a PPARγ or a γ-secretase) per unit dose of the derivativeadministered as compared to the amount of the non-derivatized NSAID thatinteracts with the same non-COX polypeptide per unit dose of thenon-derivatized NSAID. In this example, because the derivative binds toCOX enzymes less strongly than the parent NSAID, on a mole-for-molebasis, more of the derivative should be available to interact withnon-COX polypeptides than would the parent NSAID.

As used herein, the terms “significantly less” and “significantlyreduced” refer to a result (for example, an amount of a product of anenzymatic reaction) that is reduced by more than the margin of errorinherent in the measurement technique, in some embodiments a decrease byabout 2 fold or greater with respect to a baseline activity (forexample, the activity of the wild type enzyme in the absence of theinhibitor), in some embodiments, a decrease by about 5 fold or greater,and in still some embodiments a decrease by about 10 fold or greater.

As used herein, the phrases “treatment effective amount”,“therapeutically effective amount”, and “treatment amount” are usedinterchangeably and refer to an amount of a therapeutic compositionsufficient to produce a measurable response (e.g., a biologically orclinically relevant response in a subject being treated). Actual dosagelevels of active ingredients in the pharmaceutical compositions of thepresently disclosed subject matter can be varied so as to administer anamount of the active compound(s) that is effective to achieve thedesired therapeutic response for a particular subject. The selecteddosage level will depend upon the activity of the therapeuticcomposition, the route of administration, combination with other drugsor treatments, the severity of the condition being treated, and thecondition and prior medical history of the subject being treated.However, it is within the skill of the art to start doses of thecompound at levels lower than required to achieve the desiredtherapeutic effect and to gradually increase the dosage until thedesired effect is achieved.

The potency of a therapeutic composition can vary, and therefore a“therapeutically effective amount” can vary. However, one skilled in theart can readily assess the potency and efficacy of a candidate modulatorof the presently disclosed subject matter and adjust the therapeuticregimen accordingly.

After review of the disclosure herein of the presently disclosed subjectmatter, one of ordinary skill in the art can tailor the dosages to anindividual subject, taking into account the particular formulation,method of administration to be used with the composition, and otherfactors. Further calculations of dose can consider subject height andweight, severity and stage of symptoms, and the presence of additionaldeleterious physical conditions. Such adjustments or variations, as wellas evaluation of when and how to make such adjustments or variations,are well known to those of ordinary skill in the art of medicine.

III. DERIVATIZATION OF NSAIDs

III.A. General Considerations

Cyclooxygenases (COXs) are the therapeutic targets of non-steroidalanti-inflammatory drugs. Indomethacin (INDO) was one of the firstnon-steroidal anti-inflammatory drugs to be characterized as afunctionally irreversible, time-dependent inhibitor, but the molecularbasis underlying this phenomenon is uncertain. In the crystal structureof INDO bound to COX-2, a small hydrophobic pocket was identified thatsurrounds the 2′ methyl group of INDO. The pocket is formed by theresidues Ala-527, Val-349, Ser-530, and Leu-531. The contribution ofthis pocket to inhibition was evaluated by altering its volume bymutagenesis of Val-349. The V349A mutation expanded the pocket andincreased the potency of INDO, whereas the V349L mutation reduced thesize of the pocket and decreased the potency of INDO. Particularlystriking was the reversibility of INDO inhibition of the V349L mutant.

NSAIDs have been found to have various activities, including the abilityto modulate the activities of cyclooxygenases (e.g., COX-1 and/orCOX-2), PPARs (e.g., PPARγ), and secretases (e.g., γ-secretase). Theability to create different derivatives of NSAIDs can be exploited todifferentially modulate the activities of these polypeptides, which canbe used to treat different diseases and disorders.

The use of an NSAID to modulate a PPAR and/or a secretase in vivo iscompromised by the presence of significant gastrointestinal toxicitiesinduced by high dosage administration of the NSAID. This effect appearsto be due to the inhibition of COX-1, resulting in a reduction in theproduction and release of cytoprotective prostaglandins in thegastrointestinal (GI) tract. One approach to reducing GI toxicity is toreduce the ability of the NSAID to bind to COX-1 and/or COX-2, yetmaintain the ability to modulate other target polypeptides.

Thus, in some embodiments, the presently disclosed subject matterprovides a method for altering the specificity of acyclooxygenase-inhibiting compound. In some embodiments, the methodcomprises (a) providing a compound having cyclooxygenase inhibitoryactivity, the compound comprising an indoleacetic acid or indenaceticacid functional group having a 2′ methyl group; and (b) replacing the 2′methyl group with a moiety selected from the group consisting ofhydrogen; halo; halomethyl, wherein at least one hydrogen of the methylgroup is substituted with a halogen; C₂ to C₆ alkyl; C₂ to C₆ branchedalkyl; and C₂ to C₆ substituted alkyl to create a derivative, whereinthe derivative substantially lacks cyclooxygenase inhibitory activity.In some embodiments of this method, the compound is a non-steroidalanti-inflammatory drug. Thus, in some embodiments, the derivative is aderivative of an NSAID, and comprises an indoleacetic acid orindeneacetic acid functional group having a hydrogen or a fluorinesubstituent at the 2′ position. Representative NSAIDs comprising anindoleacetic acid or indenacetic acid functional group include, but arenot limited to indomethacin and sulindac, as well as pharmaceuticallyacceptable salts thereof and combinations thereof.

The structures of indomethacin and sulindac are presented below.

The 2′ methyl groups are shown attached to the indoleacetic acid andindenacetic acid functional groups, respectively. These 2′ methyl groupsplay an important role in binding of these NSAIDs to COX enzymes, andthus removal of the 2′ methyl groups to form 2-Des-methyl derivativescan be used to reduce the ability of the 2-Des-methyl derivatives tobind to COX enzymes without negatively affecting the ability of thederivatives to bind to and/or interact with PPARs, secretases, and othertarget polypeptides. Accordingly, in some embodiments, the derivative isselected from the group consisting of 2-Des-methylindomethacin, eindenicacid sulfide, eindenic acid sulfoxide, and eindenic acid sulfone. Insome embodiments, the derivative is eindenic acid sulfide.

Additionally, all positions corresponding to positions where hydrogen ispresent in the parent compounds can also be derivatized, and should beviewed as R groups (e.g., R¹, R², R³, etc.). As such, in someembodiments a generic structure for the presently disclosed derivativesis presented as Formula I:

wherein

-   -   R¹ is selected from the group consisting of hydrogen, halo, CF₃;        SCH₃; SOCH₃; SO₂CH₃; SO₂NH₂; C₁ to C₆ alkyl, branched alkyl, or        substituted alkyl; C₁ to C₆ alkoxy, branched alkoxy, or        substituted alkoxy; C₁ to C₆ alkylcarboxylic acid, branched        alkylcarboxylic acid, or substituted alkylcarboxylic acid; and        CH₂N₃;    -   R² is selected from the group consisting of hydrogen, halo, CF₃;        SCH₃; SOCH₃; SO₂CH₃; SO₂NH₂; CONH₂; C₁ to C₆ alkyl, branched        alkyl, or substituted alkyl; C₁ to C₆ alkoxy, branched alkoxy,        or substituted alkoxy; benzyloxy; C₁ to C₆ alkylcarboxylic acid,        branched alkylcarboxylic acid, or substituted alkylcarboxylic        acid; and CH₂N₃;    -   R³ and R⁴ are each independently selected from the group        consisting of hydrogen; halo; CF₃; C₁ to C₆ alkyl, branched        alkyl, or substituted alkyl; C₁ to C₆ alkoxy, branched alkoxy,        or substituted alkoxy; aryl; substituted aryl; benzyloxy; SCH₃;        SOCH₃; SO₂CH₃; and SO₂NH₂;    -   R⁵ is selected from the group consisting of hydrogen, C₁ to C₆        alkyl, branched alkyl, or substituted alkyl, and ═O;    -   R⁶ is selected from the group consisting of hydrogen; C₁ to C₆        alkyl, branched alkyl, or substituted alkyl; C₁ to C₆ alkoxy,        branched alkoxy, or substituted alkoxy; benzyloxy; C₁ to C₆        alkylcarboxylic acid, branched alkylcarboxylic acid, or        substituted alkylcarboxylic acid; and the following structure:

-   -   wherein        -   Ar is cyclohexyl or phenyl;        -   R⁷ is hydrogen; C₁ to C₆ alkyl, branched alkyl, or            substituted alkyl;        -   R⁸ is hydrogen, halo, C₁ to C₆ alkyl, branched alkyl, and            substituted alkyl; C₁ to C₆ alkoxy, branched alkoxy, and            substituted alkoxy; C₁ to C₆ alkylcarboxylic acid, branched            alkylcarboxylic acid, or substituted alkylcarboxylic acid;            amino; nitro; CF₃; bromoacetamidyl; benzoyl; or            2-phenyl-oxiranyl;        -   X is O or NR⁹, wherein R⁹ is hydrogen or alkyl; and        -   m, n, s, and t are each individually 0, 1, 2, 3, 4, or 5;    -   Y is selected from the group consisting of hydrogen, halo, CF₃,        and C₂ to C₆ alkyl, branched alkyl, or substituted alkyl;    -   A is selected from the group consisting of carbon and nitrogen;    -   p and q are both individually 0, 1, 2, 3, or 4;    -   the bond between the carbon bound to R⁵ and the indene ring and        is a single bond or a double bond; and    -   the six-membered ring to which R¹ is bound is cyclohexyl or        phenyl.

Continuing with reference to Formula I, in some embodiments, R¹ isselected from the group consisting of halo, C₁ to C₆ alkyl or branchedalkyl, SCH₃, SOCH₃, SO₂CH₃, and SO₂NH₂; R² is selected from the groupconsisting of hydrogen; halo; C₁ to C₆ alkyl or branched alkyl; C₁ to C₆alkoxy or branched alkoxy; benzyloxy; SCH₃; SOCH₃; SO₂CH₃; SO₂NH₂; andCONH₂; R³ and R⁴ are each independently selected from the groupconsisting of hydrogen, C₁ to C₆ alkyl or branched alkyl, and halo; R⁵is selected from the group consisting of hydrogen, C₁ to C₆ alkyl orbranched alkyl, and carbonyl; R⁶ is selected from the group consistingof C₁ to C₆ alkylcarboxylic acid and branched C₁ to C₆ alkylcarboxylicacid; Y is selected from the group consisting of hydrogen, halo, and C₂to C₆ alkyl or branched alkyl; A is selected from the group consistingof carbon and nitrogen; and the bond between the carbon bound to R⁵ andthe indene ring is a single bond or a double bond.

In some embodiments, a generic structure for the presently disclosedderivatives is presented as Formula Ia or Formula Ib:

wherein:

-   -   R² is selected from the group consisting of hydrogen, halo, CF₃;        SCH₃; SOCH₃; SO₂CH₃; SO₂NH₂; CONH₂; C₁ to C₆ alkyl, branched        alkyl, or substituted alkyl; C₁ to C₆ alkoxy, branched alkoxy,        or substituted alkoxy; benzyloxy; C₁ to C₆ alkylcarboxylic acid,        branched alkylcarboxylic acid, or substituted alkylcarboxylic        acid; and CH₂N₃;    -   R⁴ is selected from the group consisting of hydrogen; halo; CF₃;        C₁ to C₆ alkyl, branched alkyl, or substituted alkyl; C₁ to C₆        alkoxy, branched alkoxy, or substituted alkoxy; aryl;        substituted aryl; benzyloxy; SCH₃; SOCH₃; SO₂CH₃; and SO₂NH₂;    -   R⁵ is selected from the group consisting of hydrogen, C₁ to C₆        alkyl, C₁ to C₆ branched alkyl, and C₁ to C₆ substituted alkyl,        and in the case of Formula Ia, ═O;    -   R⁶ is selected from the group consisting of hydrogen; C₁ to C₆        alkyl, branched alkyl, or substituted alkyl; C₁ to C₆ alkoxy,        branched alkoxy, or substituted alkoxy; benzyloxy; C₁ to C₆        alkylcarboxylic acid, branched alkylcarboxylic acid, or        substituted alkylcarboxylic acid; and the following structure:

-   -   wherein        -   Ar is cyclohexyl or phenyl;        -   R⁷ is hydrogen; C₁ to C₆ alkyl, branched alkyl, or            substituted alkyl;        -   R⁸ is hydrogen, halo, C₁ to C₆ alkyl, branched alkyl, and            substituted alkyl; C₁ to C₆ alkoxy, branched alkoxy, and            substituted alkoxy; C₁ to C₆ alkylcarboxylic acid, branched            alkylcarboxylic acid, or substituted alkylcarboxylic acid;            amino; nitro; CF₃; bromoacetamidyl; benzoyl; or            2-phenyl-oxiranyl;        -   X is O or NR⁹, wherein R⁹ is hydrogen or alkyl; and        -   m, n, and t are each individually 0, 1, 2, 3, 4, or 5;    -   R¹⁵ is selected from the group consisting of cyclohexyl and an        aromatic substituent, wherein the aromatic substituent comprises        an aryl, a heteroaryl, and singly or multiply substituted        derivatives thereof;    -   Y is selected from the group consisting of hydrogen; halo;        halomethyl, wherein at least one hydrogen of the methyl group is        substituted with a halogen; C₂ to C₆ alkyl; C₂ to C₆ branched        alkyl; and C₂ to C₆ substituted alkyl; and    -   q is 0, 1, 2, 3, or 4.

In some embodiments, R¹⁵ comprises an aryl comprising multiple aromaticrings that are fused together or covalently linked. In some embodiments,the multiple aromatic rings are covalently linked by a moiety selectedfrom the group consisting of an alkylene moiety, a carbonyl, oxygen,diphenylether, and nitrogen. In some embodiments, the multiple aromaticrings are selected from the group consisting of naphthyl, biphenyl,diphenylether, diphenylamine, and benzophenone.

In some embodiments, the derivative is selected from the groupconsisting of 2-Des-methylindomethacin, eindenic acid sulfide, eindenicacid sulfoxide, and eindenic acid sulfone. In some embodiments, thederivative is eindenic acid sulfide.

In some embodiments, the compound is a compound of Formula Ib, wherein:

-   -   R² is selected from the group consisting of hydrogen, halo, CF₃;        SCH₃; SOCH₃; SO₂CH₃; SO₂NH₂; CONH₂; C₁ to C₆ alkyl, branched        alkyl, or substituted alkyl; C₁ to C₆ alkoxy, branched alkoxy,        or substituted alkoxy; benzyloxy; C₁ to C₆ alkylcarboxylic acid,        branched alkylcarboxylic acid, or substituted alkylcarboxylic        acid; and CH₂N₃;    -   R⁴ is selected from the group consisting of hydrogen; halo; CF₃;        C₁ to C₆ alkyl, branched alkyl, or substituted alkyl; C₁ to C₆        alkoxy, branched alkoxy, or substituted alkoxy; aryl;        substituted aryl; benzyloxy; SCH₃; SOCH₃; SO₂CH₃; and SO₂NH₂;    -   R⁵ is selected from the group consisting of hydrogen, C₁ to C₆        alkyl, C₁ to C₆ branched alkyl, and C₁ to C₆ substituted alkyl;    -   R⁶ is selected from the group consisting of C₁ to C₆        alkylcarboxylic acid, branched alkylcarboxylic acid, and        substituted alkylcarboxylic acid;    -   R¹⁵ is selected from the group consisting of cyclohexyl and an        aromatic substituent, wherein the aromatic substituent comprises        an aryl, a heteroaryl, and singly or multiply substituted        derivatives thereof;    -   Y is selected from the group consisting of hydrogen; halo;        halomethyl, wherein at least one hydrogen of the methyl group is        substituted with a halogen; C₂ to C₆ alkyl; C₂ to C₆ branched        alkyl; and C₂ to C₆ substituted alkyl; and    -   q is 0, 1, 2, 3, or 4.

In some embodiments, R¹⁵ comprises an aryl comprising multiple aromaticrings that are fused together or covalently linked. In some embodiments,the multiple aromatic rings are covalently linked by a moiety selectedfrom the group consisting of an alkylene moiety, a carbonyl, oxygen,diphenylether, and nitrogen. In some embodiments, the multiple aromaticrings are selected from the group consisting of naphthyl, biphenyl,diphenylether, diphenylamine, and benzophenone.

In some embodiments, R² is selected from the group consisting of halo,C₁ to C₆ alkyl or branched alkyl, SCH₃, SOCH₃, SO₂CH₃, and SO₂NH₂. Insome embodiments, R² is F.

In some embodiments, R¹⁵ is in the E orientation with respect to theindene ring. In some embodiments, R¹⁵ can be selected from the groupincluding, but not limited to:

In derivatizing the R groups, each R group can be independentlyselected, such that any number of R groups (i.e., from zero R groups toall R groups present in a structure) can be derivatized. The term“independently selected” is used herein to indicate that the R groups,e.g., R¹, R², R³, etc. can be identical or different (e.g., R¹, R² andR³ can all be substituted alkyls, or R¹ and R⁴ can be a substitutedalkyl and R³ can be an aryl, etc.). Moreover, “independently selected”means that in a multiplicity of R groups with the same name, each groupcan be identical to or different from each other (e.g., one R³ can be analkyl, while another R³ group in the same compound can be aryl; one R⁴group can be H, while another R⁴ group in the same compound can bealkyl, etc.).

A named R group will generally have the structure that is recognized inthe art as corresponding to R groups having that name. For the purposesof illustration, representative R groups as enumerated above are definedherein. These definitions are intended to supplement and illustrate, notpreclude, the definitions known to those of skill in the art.

As used herein, the term “alkyl” means C₁₋₁₀ inclusive (i.e., carbonchains comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms; also,in some embodiments, C₁₋₆ inclusive) linear, branched, or cyclic,saturated or unsaturated (i.e., alkenyl and alkynyl)hydrocarbon chains,including for example, methyl, ethyl, propyl, isopropyl, butyl,isobutyl, tert-butyl, pentyl, hexyl, ethenyl, propenyl, butenyl,pentenyl, hexenyl, butadienyl, propynyl, butynyl, pentynyl, hexynyl, andallenyl groups.

The alkyl group can be optionally substituted with one or more alkylgroup substituents which can be the same or different, where “alkylgroup substituent” includes alkyl, halo, aryl, arylamino, acyl, hydroxy,aryloxy, alkoxyl, alkylthio, arylthio, aralkyloxy, aralkylthio, carboxy,alkoxycarbonyl, oxo and cycloalkyl. In this case, the alkyl can bereferred to as a “substituted alkyl”. Representative substituted alkylsinclude, for example, benzyl, trifluoromethyl, and the like. There canbe optionally inserted along the alkyl chain one or more oxygen, sulfuror substituted or unsubstituted nitrogen atoms, wherein the nitrogensubstituent is hydrogen, alkyl (also referred to herein as“alkylaminoalkyl”), or aryl. Thus, the term “alkyl” can also includeesters and amides. “Branched” refers to an alkyl group in which an alkylgroup, such as methyl, ethyl, or propyl, is attached to a linear alkylchain.

The term “aryl” is used herein to refer to an aromatic substituent,which can be a single aromatic ring or multiple aromatic rings that arefused together, linked covalently, or linked to a common group such as amethylene or ethylene moiety. The common linking group can also be acarbonyl as in benzophenone or oxygen as in diphenylether or nitrogen indiphenylamine. The aromatic ring(s) can include phenyl, naphthyl,biphenyl, diphenylether, diphenylamine, and benzophenone among others.In particular embodiments, the term “aryl” means a cyclic aromaticcomprising about 5 to about 10 carbon atoms, including 5 and 6-memberedhydrocarbon and heterocyclic aromatic rings. As used herein, the term“aryl” also encompasses “heteroaryl” (i.e., aryl groups containing ringatoms other than carbon). Also, the term “aryl” can also included estersand amides related to the underlying aryl group.

An aryl group can be optionally substituted with one or more aryl groupsubstituents which can be the same or different, where “aryl groupsubstituent” includes alkyl, aryl, aralkyl, hydroxy, alkoxyl, aryloxy,aralkoxyl, carboxy, acyl, halo, nitro, alkoxycarbonyl, aryloxycarbonyl,aralkoxycarbonyl, acyloxyl, acylamino, aroylamino, carbamoyl,alkylcarbamoyl, dialkylcarbamoyl, arylthio, alkylthio, alkylene and—NR′R″, where R′ and R″ can be each independently hydrogen, alkyl, aryland aralkyl. In this case, the aryl can be referred to as a “substitutedaryl”.

Specific examples of aryl groups include but are not limited tocyclopentadienyl, phenyl, furan, thiophene, pyrrole, pyran, pyridine,imidazole, isothiazole, isoxazole, pyrazole, pyrazine, pyrimidine, andthe like.

The term “alkoxy” is used herein to refer to the —OZ¹ radical, where Z¹is selected from the group consisting of alkyl, substituted alkyl,cycloalkyl, substituted cycloalkyl, heterocycloalkyl, substitutedheterocycloalkyl, silyl groups and combinations thereof as describedherein. Suitable alkoxy radicals include, for example, methoxy, ethoxy,benzyloxy, t-butoxy, etc. A related term is “aryloxy” where Z¹ isselected from the group consisting of aryl, substituted aryl,heteroaryl, substituted heteroaryl, and combinations thereof. Examplesof suitable aryloxy radicals include phenoxy, substituted phenoxy,2-pyridinoxy, 8-quinalinoxy, and the like.

The term “amino” is used herein to refer to the group —NZ¹Z², where eachof Z¹ and Z² is independently selected from the group consisting ofhydrogen, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl,heterocycloalkyl, substituted heterocycloalkyl, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkoxy, aryloxy, silyl andcombinations thereof. Additionally, the amino group can be representedas —N⁺Z¹Z²Z³, with the previous definitions applying and Z³ being eitherH or alkyl.

As used herein, the term “acyl” refers to an organic acid group whereinthe —OH of the carboxyl group has been replaced with another substituent(i.e., as represented by RCO—, wherein R is an alkyl or an aryl group asdefined herein). As such, the term “acyl” specifically includes arylacylgroups, such as an acetylfuran and a phenacyl group. Specific examplesof acyl groups include acetyl and benzoyl.

“Aroyl” means an aryl-CO— group wherein aryl is as previously described.Exemplary aroyl groups include benzoyl and 1- and 2-naphthoyl.

“Cyclic” and “cycloalkyl” refer to a non-aromatic mono- or multicyclicring system of about 3 to about 10 carbon atoms, e.g., 3, 4, 5, 6, 7, 8,9, or 10 carbon atoms. The cycloalkyl group can be optionally partiallyunsaturated. The cycloalkyl group also can be optionally substitutedwith an alkyl group substituent as defined herein, oxo, and/or alkylene.There can be optionally inserted along the cyclic alkyl chain one ormore oxygen, sulfur, or substituted or unsubstituted nitrogen atoms,wherein the nitrogen substituent is hydrogen, lower alkyl, or aryl, thusproviding a heterocyclic group. Representative monocyclic cycloalkylrings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, andcycloheptyl. Multicyclic cycloalkyl rings include adamantyl,octahydronaphthyl, decalin, camphor, camphane, and noradamantyl.

“Aralkyl” refers to an aryl-alkyl-group wherein aryl and alkyl are aspreviously described. Exemplary aralkyl groups include benzyl,phenylethyl, and naphthylmethyl. Similarly, the term “alkylaryl” refersto an alkyl-aryl-group, wherein aryl and alkyl are as previouslydescribed. As such, the terms “aralkyl” and “alkylaryl” can be usedinterchangeably, although in some instances the use of one term versusthe other is intended to express the order of a group in a chemicalstructure when read from left-to-right. By way of example, an“ethylphenyl” substituent might be distinguished from a “phenylethyl”substituent in that in the former case, the ethyl moiety is bound to themain body of the molecule while in the latter it would be the phenylmoiety that is bound to the main body of the molecule.

“Aralkyloxyl” refers to an aralkyl-O— group wherein the aralkyl group isas previously described. An exemplary aralkyloxyl group is benzyloxyl.

“Dialkylamino” refers to an —NRR′ group wherein each of R and R′ isindependently an alkyl group as previously described. Exemplaryalkylamino groups include ethylmethylamino, dimethylamino, anddiethylamino.

“Alkoxycarbonyl” refers to an alkyl-O—CO— group. Exemplaryalkoxycarbonyl groups include methoxycarbonyl, ethoxycarbonyl,butyloxycarbonyl, and t-butyloxycarbonyl.

“Aryloxycarbonyl” refers to an aryl-O—CO— group. Exemplaryaryloxycarbonyl groups include phenoxy- and naphthoxy-carbonyl.

“Aralkoxycarbonyl” refers to an aralkyl-O—CO— group. An exemplaryaralkoxycarbonyl group is benzyloxycarbonyl.

“Carbamoyl” refers to an H₂N—CO— group.

“Alkylcarbamoyl” refers to a R′RN—CO— group wherein one of R and R′ ishydrogen and the other of R and R′ is alkyl as previously described.

“Dialkylcarbamoyl” refers to a R′RN—CO— group wherein each of R and R′is independently alkyl as previously described.

“Acyloxyl” refers to an acyl-O— group wherein acyl is as previouslydescribed.

“Acylamino” refers to an acyl-NH— group wherein acyl is as previouslydescribed.

“Aroylamino” refers to an aroyl-NH— group wherein aroyl is as previouslydescribed.

The term “amino” refers to the —NH₂ group.

The term “carbonyl” refers to the —(C═O)— group.

The term “carboxyl” refers to the —COOH group.

The terms “halo”, “halide”, or “halogen” as used herein refer to fluoro,chloro, bromo, and iodo groups.

The term “halomethyl” refers to a methyl group wherein at least onehydrogen has been substituted with a halogen.

The term “hydroxyl” refers to the —OH group.

The term “hydroxyalkyl” refers to an alkyl group substituted with an —OHgroup.

The term “mercapto” refers to the —SH group.

The term “nitro” refers to the —NO₂ group.

The term “oxo” refers to a compound described previously herein whereina carbon atom is replaced by an oxygen atom.

The term “thio” refers to a compound described previously herein whereina carbon or oxygen atom is replaced by a sulfur atom.

The term “sulfate” refers to the —SO₄ group.

A “heteroatom”, as used herein, is an atom other than carbon. Exemplaryheteroatoms are heteroatoms selected from the group consisting of N, O,P, S, Si, B, Ge, Sn, and Se. In some embodiments, a heteroatom is N. Insome embodiments a heteroatom is O. In some embodiments, a heteroatom isS.

As shown in Formula I, “A” depicts a carbon or a nitrogen.

A dashed line representing a bond in a structure indicates that the bondcan either be present or absent in the structure. Thus, the dashed bondin Formula I that links the A atom to the carbon atom to which R⁵ bindsindicates that this bond can be a single bond or a double bond. The sameis true for the dashed bond depicted inside the six-membered ring towhich R¹ is bound in Formula I. For the six-membered ring, theindividual bonds can all be single, double, or a mixture of the two(e.g., the six-membered ring could be a cyclohexane ring, a benzenering, or a ring with any combination of single and/or double bonds).

As used herein, the term “eindenic acid” refers to the followingstructure:

Compounds having the general structure of eindenic acid as depicted inFormula V can be generated by 2′-desmethylation of sulindac, sulindacsulfide, etc. While the present co-inventors do not wish to berestricted to any particular theory of operation, they have observedthat the double bond of the p-substituted benzylidene moiety, which isin a Z-orientation in sulindac, can adopt an E-orientation when the2′-methyl group of sulindac is modified to a hydrogen or a fluorine.Thus, the term “eindenic acid” refers to an “E form indenacetic acid” toreflect the fact that in some embodiments this double bond is in theE-orientation. However and as indicated hereinabove, each chemicalformula or name disclosed herein encompasses all optical isomers andstereoisomers, as well as racemic mixtures where such isomers andmixtures exist. Thus, eindenic acids can adopt either the E-orientationor the Z-orientation. In some embodiments, R² and Y are defined asbefore and R⁹ and R¹⁰ are each independently selected from the groupconsisting of C₁ to C₆ alkyl, C₁ to C₆ branched alkyl, and substituted(for example, halogen-substituted) or unsubstituted aryl.

In addition to modification of the 2′ methyl position, several NSAIDshave carboxylic acid groups that can be modified. In some embodiments,the carboxylic acid moiety of indomethacin or sulindac is derivatized toan amide. In some embodiments, an amide derivative has the followinggeneral formula:

In some embodiments, R* is selected from the group consisting of aryl,alkylaryl, branched alkylaryl, and substituted aryl, wherein thesubstituted aryl comprises one or more substituents selected from thegroup consisting of halo, amino, nitro, alkoxy, hydroxyl, CF₃,haloacetamidyl (e.g. bromoacetamidyl), benzoyl, and 2-phenyl-oxiranyl.In some embodiments, the amide derivative has the following generalformula:

wherein R¹² is selected from the group consisting of phenyl-SOCH₃,phenyl-SO₂CH₃, phenyl, phenyl methyl ester, phenyl-COOH, phenyl-halo,and C₃ to C₆ cycloalkyl. Representative amide derivatives are presentedin Table 1.

TABLE 1 Representative Amide Derivatives

Com- pound R¹¹ R¹²  2

 3

 4

 5

 6

 7

 8

 9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

In some embodiments, a carboxylic acid moiety present on the compound isderivatized to an ester. In some embodiments, the ester derivative hasthe following general formula:

wherein R² is defined as above and R¹³ is defined as in Table 2. In someembodiments, the ester derivative has the following formula:

TABLE 2 Representative Ester Derivatives

Compound R² R¹³ R¹⁴ 39 SO₂CH₃ CH₂CH₃

40 OCH₃ CH₃

These and other representative derivatives of indomethacin and sulindacare presented in Table 3.

TABLE 3 Representative Derivatives Compound Formula No. Structure Mol.Wt. 1

C₁₈H₁₄ClNO₄ 343.76 2

C₂₅H₂₀FNOS 401.5 3

C₂₆H₂₂FNOS 415.52 4

C₂₇H₂₄FNOS 429.55 5

C₂₈H₂₆FNOS 443.58 6

C₂₇H₂₄FNOS 429.55 7

C₂₇H₂₄FNOS 429.55 8

C₂₇H₂₄FNOS 429.55 9

C₂₅H₂₁FN₂OS 416.51 10

C₂₅H₁₈ClF₂NOS 453.93 11

C₂₆H₂₂FNO₂S 431.52 12

C₂₆H₂₃FN₂OS 430.54 13

C₂₆H₂₃FN₂OS 430.54 14

C₂₆H₂₃FN₂OS 430.54 15

C₂₆H₂₂FNO₂S 431.52 16

C₂₆H₂₂FNO₂S 431.52 17

C₂₇H₂₄FNOS 429.55 18

C₂₇H₂₄FNOS 429.55 19

C₂₇H₂₄FNOS 429.55 20

C₂₆H₂₁ClFNOS 449.97 21

C₂₆H₂₁ClFNOS 449.97 22

C₂₆H₂₁BrFNOS 494.42 23

C₂₆H₂₁BrFNOS 494.42 24

C₂₇H₂₁F₄NOS 483.52 25

C₂₇H₂₁F₄NOS 483.52 26

C₂₆H₂₁FN₂O₃S 460.52 27

C₂₆H₂₁FN₂O₃S 460.52 28

C₂₆H₂₁FN₂O₃S 460.52 29

C₃₄H₂₇FN₄O₂ 542.6 30

C₃₃H₂₆FNO₂S 519.63 31

C₂₈H₂₃BrFN₅O₂ 560.42 32

C₂₆H₂₂FNO₂S 431.52 33

C₂₆H₂₂FNO₃S 447.52 34

C₂₅H₂₀FNO 369.43 35

C₂₆H₂₂FNO₂ 399.46 36

C₁₉H₁₃FO₄ 324.30 37

C₁₈H₁₂BrFO₂ 359.19 38

C₂₅H₂₆FNO 375.48 39

C₂₀H₁₈ClNO₅S 419.88 40

C₁₉H₁₆ClNO₄ 357.79 41

C₁₈H₁₆BrNO₄S 422.29 42

C₁₉H₁₅FO₂S 326.38 43

C₁₇H₁₄BrNO₂ 344.20 44

C₁₁H₁₁NO₄S 253.27 45

C₂₀H₁₈ClNO₄ 371.81 46

C₁₉H₁₅FO₄S 358.38 47

C₂₅H₂₁ClN₂O₃ 432.90 48

C₁₉H₁₅FO₃S 342.38 49

C₂₆H₂₈FNOS 421.57 50

C₂₁H₂₀FNOS 353.45 51

C₂₇H₂₂FNO₃S 459.53 52

C₂₃H₂₂FNO₂S 395.49 53

C₁₉H₁₆FNO₂S 308.39 54

C₂₆H₂₃NOS 397.53 55

C₁₈H₁₆FNO 281.32 56

C₁₈H₁₃FO₂ 280.29 57

C₂₅H₁₉BrFNO 448.33 58

C₁₉H₁₄FN₃O₂ 335.33 59

C₃₃H₂₅FN₄O₂ 528.58 60

C₂₆H₂₀FNO₃ 413.44 61

C₃₄H₂₈FNO₂S 533.67 62

C₁₈H₁₉FO₂ 286.14 63

C₂₆H₂₁FN₄O 424.47 64

C₂₈H₂₄BrFN₂O₂S 551.48 65

C₂₃H₁₇FN₄O₄ 432.4 66

C₃₃H₂₄FNO₄ 517.55 67

C₂₃H₁₈FNO₄S 423.46 68

C₂₀H₁₈ClN₃O₆ 431.83 69

C₃₂H₂₅ClN₂O₄ 537 70

C₁₈H₁₂BrFO₂ 359.19 71

C₁₉H₁₅FO₂ 294.32 72

C₁₉H₁₃F₄O₂ 349.30 73

C₂₀H₁₃FO₂ 304.31 74

C₁₉H₁₅FO₃ 310.32 75

C₂₂H₂₁FO₃ 352.42 76

C₁₉H₁₂F₄O₂S 380.36 77

C₂₂H₁₅FO₂ 330.35 78

C₂₂H₁₅FO₂ 330.35 79

C₂₂H₁₄F₂O₂ 348.34 80

C₂₁H₁₄FNO₂ 331.34 81

C₂₄H₁₇FO₂ 356.39 82

C₂₅H₁₉FO₂S 402.48 83

C₂₄H₁₅F₃O₂ 424.43

III.B. Modulation of PPARs

Diabetes mellitus is a condition in which the glucose homeostasis of asubject becomes unbalanced and leads to a hyperglycemic systemiccondition. There are two forms of the diabetic condition, Type I andType II. Type I diabetes usually occurs in individuals underapproximately 20 years of age, is insulin-dependent, is commonlyaccompanied by ketoacidosis and represents about 10% of the diabeticpopulation. Type II diabetes affects approximately 5 percent of theadult American population and represents about 90% of the diabeticpopulation. Type II diabetes is commonly associated with obesity,usually occurs in individuals over approximately 40 years of age and isnon-insulin dependent. A subset of type II diabetes can occur in youngerindividuals and is referred to as maturity onset diabetes of the young(MODY).

PPARs, particularly PPARγ, have been implicated in mediatingdifferentiation of adipocytes and regulating fat metabolism.Additionally, PPARγ has been associated with various pathologicalconditions related to atherosclerosis, inflammation, obesity, diabetes,the immune response, and ageing. See Kersten et al., 2000; Celi &Shuldiner, 2002.

In some embodiments, the presently disclosed subject matter provides amethod of modulating the activity of a PPAR (e.g., PPARγ). In thisembodiment, a treatment effective amount of a derivative of thepresently disclosed subject matter is administered to a subject having aPPAR, whereby the activity of the PPAR is modulated.

III.C. Modulation of Cell Growth

Peroxisome proliferators-activated receptors (PPARS) have beenassociated with various pathological conditions related toatherosclerosis, inflammation, obesity, diabetes, the immune response,and ageing. Activation of one particular member of this family ofreceptors, PPARγ, by cyclopentenone prostaglandins (PGs) such as15-deoxy-Δ^(12,14)-prostaglandin J₂ (15d-PGJ₂), causesanti-proliferation, apoptosis, differentiation, and anti-inflammatoryresponses in certain types of cancer cells.

In some embodiments, the presently disclosed subject matter provides amethod of modulating the activity of a PPAR (e.g., PPARγ). In thisembodiment, a treatment effective amount of a derivative of thepresently disclosed subject matter is administered to a subject having aPPAR, whereby the activity of the PPAR is modulated.

III.D. Modulation of Secretases

As discussed in more detail hereinabove, secretases are involved in theprocessing of Aβ peptide, including the generation of Aβ42, thepurported etiologic agent in Alzheimer's disease. In some embodiments,the presently disclosed subject matter provides a method of modulatingthe activity of a secretase (e.g., γ-secretase). In this embodiment, atreatment effective amount of a derivative of the presently disclosedsubject matter is administered to a subject having a secretase, wherebythe activity of the secretase is modulated.

IV. TREATMENT METHODS

IV.A. Subjects

The presently disclosed subject matter also provides a method fortreating a disease in a subject, wherein the disease is selected fromthe group consisting of a cancer, a neurodegenerative disease, anddiabetes, the method comprising administering to the subject a treatmenteffective amount of a derivative of a compound, wherein the compoundcomprises a cyclooxygenase inhibitor comprising an indoleacetic acid orindenacetic acid functional group having a 2′ methyl group and thederivative substantially lacks cyclooxygenase inhibitory activity as aresult of modifying the 2′ methyl group to a moiety selected from thegroup consisting of hydrogen, halo, and C₂ to C₆ alkyl or branchedalkyl.

As used herein, the phrase “treating a disease in a subject” refers toboth intervention designed to ameliorate the symptoms of causes of thedisease in a subject (e.g., after initiation of the disease process) aswell as to interventions that are designed to prevent the disease fromoccurring in the subject. Stated another way, the terms “treating” andgrammatical variants thereof are intended to be interpreted broadly toencompass meanings that refer to reducing the severity of and/or tocuring a disease, as well as meanings that refer to prophylaxis. In thislatter respect, “treating” refers to “preventing” or otherwise enhancingthe ability of the subject to resist the disease process.

The subjects treated in the presently disclosed subject matter in itsmany embodiments is desirably a human subject, although it is to beunderstood that the principles of the presently disclosed subject matterindicate that the presently disclosed subject matter is effective withrespect to invertebrate and to all vertebrate animals, includingmammals, which are intended to be included in the term “subject”.Moreover, a mammal is understood to include any mammalian species inwhich treatment or prevention of a disease is desirable, particularlyagricultural and domestic mammalian species. For example, the presentlydisclosed subject matter is applicable to the treatment of livestock.

The methods of the presently disclosed subject matter are particularlyuseful in the treatment of warm-blooded vertebrates. Thus, the presentlydisclosed subject matter concerns mammals and birds.

More particularly provided is the treatment of mammals such as humans,as well as those mammals of importance due to being endangered (such asSiberian tigers), of economic importance (animals raised on farms forconsumption by humans) and/or social importance (animals kept as pets orin zoos) to humans, for instance, carnivores other than humans (such ascats and dogs), swine (pigs, hogs, and wild boars), ruminants (such ascattle, oxen, sheep, giraffes, deer, goats, bison, and camels), andhorses. Also provided is the treatment of birds, including the treatmentof those kinds of birds that are endangered, kept in zoos, as well asfowl, and more particularly domesticated fowl, i.e., poultry, such asturkeys, chickens, ducks, geese, guinea fowl, and the like, as they arealso of economic importance to humans. Thus, contemplated is thetreatment of livestock, including, but not limited to, domesticatedswine (pigs and hogs), ruminants, horses, poultry, and the like.

IV.B. Formulation

The compositions of the presently disclosed subject matter comprise insome embodiments a composition that includes a carrier, particularly apharmaceutically acceptable carrier. Any suitable pharmaceuticalformulation can be used to prepare the compositions for administrationto a subject.

For example, suitable formulations can include aqueous and non-aqueoussterile injection solutions that can contain anti-oxidants, buffers,bacteriostatics, bactericidal antibiotics and solutes which render theformulation isotonic with the bodily fluids of the intended recipient;and aqueous and non-aqueous sterile suspensions which can includesuspending agents and thickening agents. The formulations can bepresented in unit-dose or multi-dose containers, for example sealedampoules and vials, and can be stored in a frozen or freeze-dried(lyophilized) condition requiring only the addition of sterile liquidcarrier, for example water for injections, immediately prior to use.Some exemplary ingredients are SDS, in one example in the range of 0.1to 10 mg/ml, in another example about 2.0 mg/ml; and/or mannitol oranother sugar, for example in the range of 10 to 100 mg/ml, in anotherexample about 30 mg/ml; and/or phosphate-buffered saline (PBS).

It should be understood that in addition to the ingredients particularlymentioned above the formulations of the presently disclosed subjectmatter can include other agents conventional in the art with regard tothe type of formulation in question. For example, sterile pyrogen-freeaqueous and non-aqueous solutions can be used.

The therapeutic regimens and compositions of the presently disclosedsubject matter can be used with additional adjuvants or biologicalresponse modifiers including, but not limited to, cytokines and otherimmunomodulating compounds.

IV.C. Administration

Administration of the compositions of the presently disclosed subjectmatter can be by any method known to one of ordinary skill in the art,including, but not limited to intravenous administration, intrasynovialadministration, transdermal administration, intramuscularadministration, subcutaneous administration, topical administration,rectal administration, intravaginal administration, intratumoraladministration, oral administration, buccal administration, nasaladministration, parenteral administration, inhalation, and insufflation.In some embodiments, suitable methods for administration of acomposition of the presently disclosed subject matter include but arenot limited to intravenous injection. The particular mode ofadministering a composition of the presently disclosed subject matterdepends on various factors, including the distribution and abundance ofcells to be treated, the compound employed, additional tissue- orcell-targeting features of the compound, and mechanisms for metabolismor removal of the compound from its site of administration.

IV.D. Dose

An effective dose of a composition of the presently disclosed subjectmatter is administered to a subject in need thereof. A “treatmenteffective amount” or a “therapeutic amount” is an amount of atherapeutic composition sufficient to produce a measurable response(e.g., a biologically or clinically relevant response in a subject beingtreated). In some embodiments, an activity that inhibits amyloidaggregate formation is measured. Actual dosage levels of activeingredients in the compositions of the presently disclosed subjectmatter can be varied so as to administer an amount of the activecompound(s) that is effective to achieve the desired therapeuticresponse for a particular subject. The selected dosage level will dependupon the activity of the therapeutic composition, the route ofadministration, combination with other drugs or treatments, the severityof the condition being treated, and the condition and prior medicalhistory of the subject being treated. However, it is within the skill ofthe art to start doses of the compound at levels lower than required toachieve the desired therapeutic effect and to gradually increase thedosage until the desired effect is achieved. The potency of acomposition can vary, and therefore a “treatment effective amount” canvary. However, using the assay methods described herein, one skilled inthe art can readily assess the potency and efficacy of a candidatecompound of the presently disclosed subject matter and adjust thetherapeutic regimen accordingly.

After review of the disclosure of the presently disclosed subject matterpresented herein, one of ordinary skill in the art can tailor thedosages to an individual subject, taking into account the particularformulation, method of administration to be used with the composition,and particular disease treated. Further calculations of dose canconsider subject height and weight, severity and stage of symptoms, andthe presence of additional deleterious physical conditions. Suchadjustments or variations, as well as evaluation of when and how to makesuch adjustments or variations, are well known to those of ordinaryskill in the art of medicine.

EXAMPLES

The following Examples provide illustrative embodiments. Certain aspectsof the following Examples are described in terms of techniques andprocedures found or contemplated by the present inventors to work wellin the practice of the embodiments. In light of the present disclosureand the general level of skill in the art, those of skill willappreciate that the following Examples are intended to be exemplary onlyand that numerous changes, modifications, and alterations can beemployed without departing from the scope of the presently disclosedsubject matter.

Example 1 Mutagenesis and Purification of mCOX-2

Site-directed mutagenesis, expression, and purification of murine COX-2(mCOX-2) nucleic acids and polypeptides were performed as described inRowlinson et al., 1999. Briefly, PCR-mediated site-directed mutagenesiswas performed on a mCOX-2 coding sequence present in a BLUESCRIPT®vector (Stratagene, La Jolla, Calif., United States of America) usingthe QUIKCHANGE® Site-Directed Mutagenesis Kit (Stratagene). The GTGcodon encoding a valine at position 335 in wild type mCOX-2 (referred toherein as Val-349 based on the numbering convention discussedhereinabove) was changed to encode an alanine, a leucine, or anisoleucine, creating three mutant nucleic acids encoding mCOX-2polypeptides referred to herein as mCOX-2^(V349A) or V349A,mCOX-2^(V349L) or V339L, and mCOX-2^(V349I) or V349I, respectively.

In order to express the mutant polypeptides, sequences containing themutagenized codons were removed from the mCOX-2-containing BLUESCRIPT®vector and subcloned into a pVL1393 baculovirus expression vector (BDBiosciences PharMingen, San Diego, Calif., United States of America)encoding mCOX-2 using the BamHI restriction site present in both themCOX-2-containing BLUESCRIPT® and pVL1393 vectors. The subcloned regionwas fully sequenced to ensure that no additional mutations wereincorporated into the expression vectors.

Wild type and mutant protein was then expressed by homologousrecombination of the mCOX-2z/pVL1393 vector with the BACULOGOLD™ vector(BD Biosciences PharMingen) in Sf9 cells (EMD Biosciences, Inc.—Novagen,Madison, Wis., United States of America). After virus amplification, 4liters of Sf9 cells (95-100% viable) were grown in TNM-FH mediumsupplemented with 10% fetal bovine serum (FBS; HyClone, Logan, Utah,United States of America), 1% L-glutamine, and 0.1% (v/v) pluronic F68and then infected with fresh viral stock. Upon reaching 65-70%viability, the 4-liter total volume was harvested by centrifugation at2500 rpm in a Sorvall RC-3B centrifuge, and the pellet was washed inice-cold phosphate-buffered saline and re-centrifuged. The final cellpellet was stored at −80° C.

Purification of wild type and mutant mCOX-2 polypeptides was performedat 4° C. in a manner similar to that described in Gierse et al., 1996.Briefly, frozen cells were resuspended to 30×10⁶ cells/ml in 80 mMTris-HCl, 2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, and 0.1 mMdiethyldithiocarbamic acid, pH 7.2. After centrifugation at 100,000 gfor 45 minutes, the pellet was resuspended using a Dounce homogenizer toa final volume of 72 ml. Solubilization of the COX protein from themembrane was initiated by the dropwise addition of 8 ml of 11% (w/v)CHAPS. After stirring for 1 hour, the sample was re-centrifuged asdescribed above and the supernatant removed and then diluted 4-fold bythe addition of 20 mM Tris-HCl, 0.4% CHAPS, 0.1 mM EDTA, and 0.1 mMdiethyldithiocarbamic acid, pH 8.0 (Buffer B). The diluted sample wasthen loaded onto a 25 ml Macro-prep High-Q ion exchange columnequilibrated with Buffer B. COX enzyme was eluted with a linear gradient(500 ml) of increasing KCl to 0.3 M. An analytical 7.5%SDS-polyacrylamide gel electrophoresis was run of candidateCOX-containing fractions to determine the fractions to pool for the gelfiltration procedure. Appropriate tubes were concentrated in an Amiconconcentrator (Amicon, Beverly, Mass., United States of America) to afinal volume of 1.5 ml. The sample was then loaded onto a 90 mlSephacryl-200 column that was pre-equilibrated with 20 mM Tris-HCl, 0.4%CHAPS, 0.15 M NaCl, pH 8.0. Fractions containing COX enzyme, asdetermined from SDS-polyacrylamide gel electrophoresis analysis, wereconcentrated to approximately 2 mg/ml and stored at −80° C. The purityof wild type and mutant COX-2 proteins was evaluated by analysis of aCoomassie-stained 7.5% SDS-polyacrylamide gel using an E-C ApparatusModel EC910 scanning densitometer (E-C Apparatus Corp., Holbrook, N.Y.,United States of America). All proteins were over 80% pure.

Example 2 Reagents and Solvents

Unlabeled arachidonic acid (AA) was purchased from Nu Chek Prep(Elysian, Minn., United States of America), and [1-¹⁴C]-AA was purchasedfrom PerkinElmer Life Sciences Inc. (Boston, Mass., United States ofAmerica). Ram seminal vesicles were purchased from Oxford BiomedicalResearch (Oxford, Mich., United States of America). Oligonucleotideswere purchased from Qiagen, Inc. (Valencia, Calif., United States ofAmerica) and all molecular biology enzymes were obtained from NewEngland Biolabs (Beverly, Mass., United States of America). Baculovirusreagents were purchased from BD Biosciences Pharmingen (San Diego,Calif., United States of America). Unless otherwise stated, all otherchemicals were obtained from Sigma/Aldrich (St. Louis, Mo., UnitedStates of America). HPLC grade solvents used for column chromatographywere obtained from Fischer Scientific (Pittsburgh, Pa., United States ofAmerica). N,N-Dimethylformamide was distilled from calcium hydride. Allother chemicals were used without further purification. Thin layerchromatography was performed on silica plates obtained from Analtech(Newark, Del., United States of America; Silica Gel 60 F₂₅₄ precoated).The plates were read by UV fluorescence (254 nm) or by staining withphosphomolybdic acid (PMA) followed by heating. Column chromatographywas performed using silica gel 200-300 mesh (Fisher Scientific).

Example 3 Instrumental Analysis

Mass spectra were obtained by electrospray ionization (ESI-MS) on aFinnigan TSQ 7000 triple-quadrupole spectrometer (Thermo Electron Corp.,Waltham, Mass., United States of America). ¹H-NMR were obtained on aBruker AC 300 NMR spectrometer (Bruker BioSpin Corporation, Billerica,Mass., United States of America) using CDCl₃ or DMSO-d₆ as the solventand tetramethylsilane (TMS) as an internal standard. All chemical shiftsare reported in parts per million (ppm) downfield from TMS and couplingconstants are reported in hertz.

Example 4 Synthesis of 2-Des-methylindomethacin

2-Des-methylindomethacin was synthesized according to the steps outlinedin FIG. 6. These steps are presented in more detail as follows:

N-Ethylidene-N′-(4-methoxy-phenyl)-hydrazone (Compound A in FIG. 6).4-Methoxyphenylhydrazine (10.34 g, 0.075 mol) was dissolved in toluene(76 mL) in a flame dried round-bottomed flask. The flask was purged withargon and cooled to 0° C. Acetaldehyde (8.4 mL, 0.15 mol) in toluene (17mL) was added dropwise and stirring at room temperature was continuedfor 30 minutes. The solution was decanted through filter paper into around-bottomed flask and concentrated in vacuo to give 11.2 g of thecrude hydrazone (Compound A in FIG. 6).

4-Chloro-benzoic acid N′-ethylidene-N-(4-methoxy-phenyl)-hydrazide(Compound B in FIG. 6). To a solution of the crude hydrazone (Compound Ain FIG. 6; 0.424 g, 2.58 mmol) in pyridine (2.2 mL) under argon at 0° C.was added 4-chlorobenzoyl chloride (0.904 g, 5.17 mmol) in one portion.The reaction mixture was stirred at room temperature for 3 hours. Water(25 mL) was added and the solution extracted with CH₂Cl₂ (3×20 mL). Thecombined organic phases were dried (MgSO₄), filtered, and concentratedin vacuo. Purification by flash chromatography (hexane/ethyl acetate3:1) afforded the title compound (Compound B in FIG. 6) as an orangesolid (0.266 g). ¹H NMR (CDCl₃) δ 7.70 (d, J=7.2 Hz, 2H), 7.36 (d, J=8.2Hz, 2H), 7.11 (d, J=8.7 Hz, 2H), 7.01 (d, J=8.5 Hz, 2H), 6.82 (d, J=5.0Hz, 1H), 3.84 (s, 3H), 1.89 (d, J=5.0 Hz, 3H); ESI-CID 325, 327(M-Na⁺[^(35/37)Cl]).

4-Chloro-benzoic acid N-(4-methoxy-phenyl)-hydrazide hydrochloride(Compound C in FIG. 6). The hydrazide (Compound B in FIG. 6; 0.166 g,0.55 mmol) was dissolved in toluene (6.6 mL) and methanol (0.33 mL) in a2 neck flask equipped with a condensor. The mixture was cooled to 0° C.and HCl gas was bubbled through for 1.5 hours. The excess gas andsolvent were removed in vacuo. The solid was swirled with toluene andfiltered to give a white solid. The solid was washed with ethyl acetateto give the hydrochloride salt (Compound C in FIG. 6) without furtherpurification (0.131 g, 76%). ¹H NMR (DMSO-d₆) δ 7.41 (m, 4H), 7.33 (d,J=8.4 Hz, 2H), 6.92 (d, J=8.9 Hz), 3.74 (s, 3H).

2-Des-methylindomethacin (Compound D in FIG. 6). The hydrochloride salt(Compound C in FIG. 6; 0.111 g, 0.35 mmol) and succinic semialdehyde(0.047 g, 0.46 mmol) were dissolved in acetic acid (AcOH; 2 mL) andheated to reflux for 4 hours. The reaction was allowed to cool to roomtemperature overnight. Water (5 mL) and CH₂Cl₂ (5 mL) were added and theorganic phase was removed. The aqueous phase was extracted with anadditional portion of CH₂Cl₂ and the combined organics were washed withwater and extracted with a saturated solution of NaHCO₃ (2×20 mL). Thecombined aqueous extracts were acidified with 15% HCl and the resultingmixture was extracted with CH₂Cl₂ (3×20 mL). The combined organics weredried (MgSO₄), filtered, and concentrated in vacuo. Recrystallization ofthe crude product (isopropanol) afforded the title compound (i.e.,[1-(4-Chloro-benzoyl)-5-methoxy-1H-indol-3-yl]-acetic acid; Compound Din FIG. 6) as a gray solid (0.058 g, 48%). ¹H NMR (DMSO-d₆) δ 8.24 (d,J=8.9 Hz, 1H), 7.82 (d, J=8.5 Hz, 2H), 7.73 (d, J=8.4 Hz, 2H), 7.39 (s,1H), 7.19 (d, J=2.3 Hz, 1H), 7.06 (dd, J=2.4, 8.9 Hz, 1H), 3.87 (s, 3H),3.73 (s, 2H); ESI-CID 342, 344 (M-H⁻[^(35/37)Cl]). Anal. Calcd forC₁₈H₁₄ClNO₄: C, 62.89; H, 4.10; N, 4.07; Cl 10.31. Found: C, 62.80; H,4.11; N, 4.06; Cl, 10.12.

Example 5 Synthesis of Eindenic Acid Sulfide

Eindenic acid sulfide was synthesized according to the steps outlined inFIG. 7. These steps are presented in more detail as follows:

6-Fluoro-indan-1-one (Compound F in FIG. 7).3-(4-Fluoro-phenyl)-propionic acid (5 g, 29.7 mmol; Compound E in FIG.7) was added to polyphosphoric acid (PPA; 65.4 g, 0.654 mol) at 50° C.The viscous mixture was heated at 90° C. for 2 hours. The syrup waspoured into ice water and stirred for 30 minutes. The aqueous mixturewas extracted with ether (3×50 mL) and the combined organics were washedwith H₂O (2×50 mL) and NaHCO₃ until neutralized. The resulting organicphase was washed with H₂O (50 mL), dried (MgSO₄), filtered, andconcentrated in vacuo. Purification using flash chromatography (7:1hexane/ethyl acetate (EtOAc)) afforded the indanone (Compound F in FIG.7) as a yellow solid (2.06 g, 46%). ¹H NMR (CDCl₃) δ 7.45 (ddd, J=0.5,4.5, 8.4 Hz, 1H), 7.39 (ddd, J=0.3, 2.6, 7.8 Hz, 1H), 7.30 (td, J=2.6,8.6 Hz, 1H), 3.12 (t, J=5.7 Hz, 2H), 2.75 (m, 2H); ESI-CID 151 (M-H⁺).

(6-Fluoro-1-hydroxy-indan-1-yl)-acetic acid ethyl ester (Compound G inFIG. 7). A solution of the indanone (Compound F in FIG. 7; 2.06 g, 13.7mmol) and ethyl bromoacetate (EBA; 3.44 g, 20.6 mmol) in benzene (10 mL)was added over a 5 minute period to activated zinc (3.77 g, 57.7 mmol)in benzene (21 mL) and ether (10 mL). A few crystals of iodine wereadded to initiate the reaction and the mixture was held at reflux. At 3hour intervals, 2 batches of zinc (1.8 g, 27.5 mmol) and ethylbromoacetate (EBA; 1.8 g, 10.8 mmol) were added and the mixture wasrefluxed overnight. The solution was cooled to room temperature andethanol (5 mL) and acetic acid (23 mL) were added. The solution waspoured into 1:1 aqueous acetic acid (100 mL) and the organic layer wasseparated. The aqueous phase was extracted with diethyl ether (Et₂O;2×25 mL) and the combined organics were washed with water, NaHCO₃,water, dried (MgSO₄), filtered, and concentrated in vacuo to give thecrude product (Compound G in FIG. 7; 3.55 g).

(6-Fluoro-3H-inden-1-yl)-acetic acid ethyl ester (Compound H in FIG. 7).The crude (6-Fluoro-1-hydroxy-indan-1-yl)-acetic acid ethyl ester(Compound G in FIG. 7; 3.55 g), p-toluene sulfonic acid.H₂O (PTSA; 5.67g, 29.8 mmol), and CaCl₂ (4.13 g, 37.2 mmol) in toluene (66 mL) wererefluxed overnight. The solution was filtered and the solid residuewashed with benzene. The combined organics were washed with water,NaHCO₃, water, dried (MgSO₄), filtered, and concentrated in vacuo.Purification using flash chromatography (13:1 hexane/EtOAc) afforded thetitle compound (Compound H in FIG. 7) as an orange solid (0.703 g). ¹HNMR (CDCl₃) δ 7.25 (m, 2H), 7.06 (m, 1H), 6.25 (d, J=2.3 Hz, 1H), 4.24(q, J=7.0 Hz, 2H), 3.32 (s, 2H), 3.03 (s, 2H), 1.33 (t, J=7.1 Hz, 3H);ESI-CID 221 (M-H⁺).

Eindenic acid sulfide (Compound I in FIG. 7). To a solution of the(6-Fluoro-3H-inden-1-yl)-acetic acid ethyl ester (Compound H in FIG. 7;0.668 g, 3.0 mmol) and p-methylthiobenzaldehyde (PMTBA; 0.508 g, 3.3mmol) in MeOH (18 mL) was added 1N NaOH (9 mL). The mixture was stirredat reflux for 2 hours. The solution was cooled, diluted with water, andextracted with ether (3×). Residual ether was blown off the aqueousphase with nitrogen and the aqueous solution acidified with 50% aceticacid. The precipitated product was filtered and washed with H₂O.Recrystallization from methanol afforded the title compound (i.e.,[6-Fluoro-3-(4-methylsulfanyl-benzylidene)-3H-inden-1-yl]-acetic acid(i.e., eidenic acid sulfide; Compound I in FIG. 7) as an orange solid(0.163 g, 17%) ¹H NMR (DMSO-d₆) δ 7.83 (dd, J=5.2, 8.4 Hz, 1H), 7.64 (d,J=8.4 Hz, 2H), 7.59 (s, 1H), 7.36 (d, J=8.4 Hz, 2H), 7.16 (dd, J=2.4,9.6 Hz, 1H), 7.13 (s, 1H), 7.06 (td, J=2.4, 9.6 Hz, 1H), 3.68 (s, 2H),2.54 (s, 3H); ESI-CID 325 (M-H⁻).

Example 6 Synthesis of a Derivative of Eindenic Acid Sulfide

To a solution of[6-Fluoro-3-(4-methylsulfanyl-benzylidene)-3H-inden-1-yl]-acetic acid(Compound I in FIG. 7; 0.02 g, 0.06 mmol) in dry CH₂Cl₂ (1.5 mL) wasadded N-(3-dimethylaminopropyl)-N′-ethyl carbodiimide (EDCI; 0.014 g,0.07 mmol), dimethylaminopyridine (DMAP; 0.75 mg, 0.006 mmol) andbenzylamine (7.9 mg, 0.07 mmol). The reaction was stirred at roomtemperature overnight. The mixture was diluted with water and extractedwith CH₂Cl₂ (2×). The combined organics were washed with H₂O, dried(MgSO₄), filtered, and concentrated in vacuo. Purification using flashchromatography (5:2 hexane/EtOAc) afforded the title compound(N-Benzyl-2-[6-fluoro-3-(4-methylsulfanyl-benzylidene)-3H-inden-1-yl]-acetamide;Compound J in FIG. 7) as an orange solid. (0.02 g, 79%).

Example 7 Cyclooxygenase Preparation and Assay COX Inhibition ScreeningAssay

PCR based site-directed mutagenesis of murine COX-2 changed the valineat position 349 (ovine COX-1 numbering) to alanine, isoleucine, orleucine as outlined in Example 1. These mutated genes were used toassemble baculoviral constructs for large-scale expression in Sf9 cells.Purification of the expressed COX-2 protein was performed throughtraditional cellular fractionation and classical column chromatography.Activity or inhibition assays were performed in 100 mM Tris-HCl buffercontaining 500 μM phenol, with hematin-reconstituted protein.Quantification of cyclooxygenase activity was performed by monitoringsubstrate (arachidonate or oxygen) consumption in a thermostattedcuvette at 37° C. using a polarographic electrode with a 5300 oxygenmonitor (Yellow Springs Instrument Co. Inc., Yellow Springs, Ohio,United States of America). All inhibitors and substrates weresolubilized in dimethyl sulfoxide (DMSO). Activity or inhibition assayswere performed in 100 mM Tris-HCl buffer containing 500 μM phenol, withhematin-reconstituted protein. Maximal reaction velocity data wereobtained from the linear portion of the oxygen uptake curves, and thedata were analyzed by nonlinear regression with Prism 4.0 (GraphPadSoftware, San Diego, Calif., United States of America).

Reactions were run with reconstituted protein at final concentrationsadjusted to give approximately 40% substrate consumption (ovine COX-1(oCOX-1)=35 nM, wild type mCOX-2=55 nM, V349A=250 nM, V3491=250 nM, andV349L=100 nM). Time-dependent inhibition reactions were performed bypre-incubating the inhibitor and enzyme for 17 minutes at 25° C.,followed by 3 minutes at 37° C. prior to the addition of 50 μM[1-¹⁴C]-AA for 30 seconds at 37° C. Assays were terminated and analyzedfor substrate consumption by thin layer chromatography as previouslydescribed (Kalgutkar et al., 2000a). All inhibitor concentrations for50% enzyme activity (IC₅₀) were determined graphically and were theaverage of at least two independent determinations.

Competitive inhibition assays were performed in a similar manner, exceptsubstrate and inhibitor were added prior the initiation of the reactionby addition of protein. The peroxidase activity of purified proteins wasmeasured by the guaiacol method as described in Markey et al., 1987. TheK_(m)'s and V_(max)'S for COX activity and the relative peroxidaseactivity of COX-2 mutants are shown in Table 4.

TABLE 4 Characterization of Val-349 mutant COXs.^(a) Peroxidase ActivityCyclooxygenase (% wild type K_(m) V_(max) ^(b) Enzyme mCOX-2) (μM) (μMof AA · min⁻¹ · mg⁻¹) wild type 100 4.2 ± 1.5 16.3 ± 1.5  mCOX-2 V349A51 13.0 ± 4.2  1.2 ± 0.1 V349I 65 9.0 ± 2.8 1.9 ± 0.2 V349L 88 15.2 ±7.3  6.0 ± 1.0 ^(a)Peroxidase activity was analyzed with the guaiacolperoxidase assay, and cyclooxygenase kinetic parameters were determinedusing at least 7 concentrations of substrate. ^(b)The cyclooxygenaseV_(max) was normalized to protein concentration and represented asspecific activity. The values are the average of three determinations ±standard error (S.E.).

Maximal reaction velocity data were obtained from the linear portion ofthe oxygen uptake curves, and the data were analyzed by nonlinearregression. Instantaneous inhibition assays were performed withsubstrate and inhibitor added prior the initiation of the reaction byaddition of protein. Time-dependent screening assays were performed bypre-incubating the inhibitor and enzyme for 17 minutes at 25° C.,followed by 3 minutes at 37° C. prior to the addition of 50 μM[1-¹⁴C]-arachidonic acid (AA) for 30 seconds at 37° C.

Assays were terminated and analyzed for substrate consumption by thinlayer chromatography. All inhibitor concentrations for 50% enzymeactivity (IC₅₀) were the average of at least two independentdeterminations. Time-dependent COX inhibition reactions werepre-incubated at 37° C. for varying lengths of time (0-30 minutes) withvarious concentrations of inhibitor. All reactions were performed with[1-¹⁴C]-AA for 30 seconds at 37° C.; reactions were terminated andanalyzed as described above.

Example 8 Time-Dependent COX Inhibition Assays

As described in Example 1, to investigate the interactions of the 2′methyl group with the methyl-binding pocket, a series of mutations weremade at position 349 in mCOX-2 to increase or decrease the volume of thepocket (Val→Ala, Ile, Leu) and the kinetics of inhibition of theseenzymes by INDO were determined. Initially, a time-dependent IC₅₀ assaywas used, in which the enzymes were pre-incubated with inhibitor for 20minutes before the addition of 50 μM AA. The COX reaction was allowed toproceed for 30 seconds before termination. The IC₅₀ values indicatedthat the potency of INDO increased when the volume of the pocketincreased (V349A) and decreased when the volume of the pocket decreased(V349I, V349L; Table 5).

TABLE 5 Time-dependent IC₅₀ determinations of INDO and DM-INDO.^(a) INDODM-INDO Enzyme (μM) (μM) V349A 0.08 >16 wild type mCOX-2 0.25 4 V349I0.45 3 V349L 4 >16 wild type oCOX-1 0.04 >16 ^(a)Values are the averageof two independent determinations

The time-dependence of inhibition of wild type and mutant COXs by INDOwas determined by adding AA (50 μM) to the various enzyme preparationsfollowing preincubation with INDO for different times. To ensure thatpseudo-first-order conditions were maintained, the enzymatic oxygenationreactions were terminated after 30 seconds to prevent extensiveconsumption of substrate. The decline of substrate conversion atdifferent INDO concentrations was plotted against the pre-incubationtimes and fit to a single exponential decay with a plateau to determinek_(obs) (FIG. 2). The time-dependent inhibition curves for wild typemCOX-2 approached 0% remaining activity. The graphs for V349A and V349Ialso approached 0% remaining activity, although V349A displayed a fasterrate of inhibition. Interestingly, V349L approached a non-zero asymptoteof nearly 20% remaining activity, which suggested that the second stepof binding was reversible (FIG. 2C). The dependence of k_(obs) on theINDO concentration for the two-step, time-dependent mechanism shown inequation (1) is represented by equation (2) (see also Timofeevski etal., 2002).

$\begin{matrix}{{{E + I}\; \underset{k_{- 1}}{\overset{k_{1}}{\rightleftharpoons}}{E - I}\underset{k_{- 2}}{\overset{k_{2}}{\rightleftharpoons}}{E - {I^{*}\mspace{14mu} K_{I}}}} = {k_{- 1}/k_{1}}} & (1) \\{k_{obs} = {\frac{k_{2} \cdot \lbrack I\rbrack}{\left( {K_{I} + \lbrack I\rbrack} \right)} + k_{- 2}}} & (2)\end{matrix}$

The rate constant k₂ represents the limiting forward rate constant forfunctionally irreversible inhibition, and K_(I) corresponds to theinhibitor concentration that yields a rate equal to half of the limitingrate. The reverse rate constant of the second step k⁻² is equal to they-intercept, and is equal to zero for compounds that displayfunctionally irreversible inhibition. For wild type mCOX-2, V349A, andV349I enzymes, the y-intercept was effectively zero, indicating that theinhibition was functionally irreversible (FIG. 2B). In contrast, thesecondary plot of data for V349L exhibited a non-zero y-intercept, whichwas equal to k⁻².

Kinetic parameters for wild type mCOX-2 (Table 6) were in good agreementwith previously reported values of K_(I) (5 μM) and k₂ (0.045 s⁻¹;Gierse et al., 1999). The values for oCOX-1 indicate that INDO displayedhigher affinity (K_(I)=1.7 μM) and a faster rate of inactivation(k₂=0.25 s⁻¹; Kulmacz & Lands, 1985). These observations are furthersupported by the higher potency of INDO towards oCOX-1 compared tomCOX-2 (Table 5). The K_(I) of V349A for INDO decreased almostfour-fold, which suggested a higher affinity of binding. This mutationalso slightly increased k₂, which corresponded to the three-foldreduction in the IC₅₀ value for INDO against the V349A enzyme (Table 5).As noted in Table 5, the V349I mutation had little effect on the kineticparameters of INDO. V349L demonstrated the greatest impact oninhibition, increasing K_(I) threefold and introducing a measurable k⁻².The slight rise in k₂ suggested a faster rate to equilibrium, which wasattributed to the emergence of k⁻² (FIG. 2C).

TABLE 6 Kinetic parameters of time-dependent inhibition by INDO.^(a)K_(I) k₂ k⁻² K⁻² ^(b) Enzyme (μM) (s⁻¹) (s⁻¹) (s⁻¹) wild type 7.9 ± 2.20.052 ± 0.005 ND ND V349A 1.9 ± 0.4 0.074 ± 0.005   ND^(c) ND V349I 5.3± 1.8 0.045 ± 0.004 ND ND V349L 26 ± 10 0.074 ± 0.013 0.008 ± 0.0020.010 ± 0.002 ^(a)Kinetic parameters ± S.E. were determined frominhibition assays. ^(b)Rate constant determined by reversibleinhibition. ^(c)Values were not detectable (ND).

Example 9 Time-Dependent COX Inhibition by DM-INDO

The experiments described in Examples 7 and 8 suggested that insertionof the 2′ methyl group of INDO into the hydrophobic pocket is animportant contributor to the time-dependence of inhibition. To furthertest this, DM-INDO (FIG. 1C) was synthesized according to the schemedepicted in FIG. 6. In the time-dependent IC₅₀ assay, DM-INDO weaklyinhibited wild type mCOX-2 and V349I but exhibited less than 20%inhibition of V349A, V349L, and wild type oCOX-1 (Table 5). The time-and concentration-dependence of inhibition of wild type enzyme and theV349L mutant exhibited a plateau of about 30% remaining activity forboth enzymes, consistent with an appreciable k⁻² (FIG. 4). Importantly,V349L required nearly 10-fold higher concentrations of DM-INDO toachieve similar levels of inhibition of wild type mCOX-2. Analysis ofthe data for inhibition of wild type enzyme by DM-INDO yielded valuesfor K_(I), k₂, and k⁻² of 26±7 μM, 0.80±0.03 s⁻¹, and 0.05±0.02 s⁻¹,respectively. Thus, the on-rate constant (k₂) is 14-fold faster forDM-INDO than INDO and the off-rate constant (k₂) is only 14-fold slowerthan the on-rate.

The kinetics of V349L inhibition by DM-INDO were not amendable toanalysis using equation (2), because the graph did not plateau. Linearregression analysis yielded a y-intercept value for k⁻² of 0.0025±0.0003s⁻¹ (Copeland, 2000). The K_(I) of DM-INDO for V349L must have been muchgreater than the equilibrium constant for the second step. Therefore,the first step in equation (1) was not saturated to allow for thetime-dependent isomerization of E-1 to E-1*(Copeland, 2000).

Example 10 Reversibility of Cox Inhibition Reversibility of COXinhibition by INDO

The reversibility of the second step in equation (1) could be directlyevaluated by the amount of enzyme activity recovered after a prolongedincubation time with substrate. To test the reversibility of INDOinhibition, wild type mCOX-2 and the three Val-349 mutants were exposedto the same conditions used for the time-dependent IC₅₀ assay. Theenzymes were pre-incubated for 20 minutes with DMSO or 10 μM INDO, priorto the addition of 50 μM AA. After addition of AA, the oxygenationreactions were allowed to proceed for varying lengths of time. As thereaction time increased, the extent of inhibition decreased if INDObinding was reversible. The time course for recovery of AA oxygenationwas fit to a single exponential (FIG. 3A). As anticipated, significantreversibility of INDO inhibition was observed for the V349L mutant butnot for wild type, V349A, or V349I enzymes. The value of k⁻² calculatedfrom the activity recovery assay (0.01 s⁻¹) corresponded closely to thek⁻² value calculated from the time-dependent inhibition assay for INDO(0.008 s⁻¹; Table 6). This relationship suggested that the reversibilityof the second step in the time-dependent mechanism is the principledeterminant of inhibition by INDO in the presence of 50 μM AA.

Reversibility of COX Inhibition by DM-INDO

The reversibility of DM-INDO inhibition was evaluated similarly to INDOusing the activity recovery assay (FIG. 3). The experiments and analysiswere performed as described above. DM-INDO displayed reversibility ofinhibition for wild type mCOX-2 and V349I, with the k⁻² values of0.023±0.001 s⁻¹ and 0.015±0.005 s⁻¹, respectively (FIG. 3B). The smallamount of activity lost for V349A and V349L by pre-incubation with 10 μMDM-INDO, was quickly recovered upon addition of 50 μM AA (FIG. 3B).

Example 11 Steady-State Quenching of COX Intrinsic Fluorescence

Fluorescence quenching experiments with INDO and DM-INDO were performedwith a Spex Fluorolog-3 spectrofluorometer (Jobin Yvon Inc., Edison,N.J., United States of America) as described in Houtzager et al., 1996.The excitation (280 nm) and emission (327 nm) bandwidths were 4 nm and 6nm respectively. Steady-state measurements were performed at 37° C. in a3.5 ml fluorescence cuvette with continuous stirring. All apo-proteinswere diluted to 200 nM, and displayed less than 2% activity of anequivalent amount of holoenzyme. Data were collected over 240 or 360seconds with 2-second integration times. The reversibility of quenchingwas analyzed in the same manner, with subsequent addition of 50 μM AA ascompetitor. The ligands were dissolved in DMSO before further dilutioninto buffer. The organic component in the buffer was below 0.4%.

Discussion of Example 11 Quenching of COX Intrinsic Fluorescence by INDOand DM-INDO

Houtzager et al. monitored inhibitor binding to COX-2 by fluorescencequenching of the apo-protein and demonstrated that the binding kineticsclosely resembled the inhibition kinetics (Houtzager et al., 1996). Thisassay provided a method to directly monitor the binding of INDO andDM-INDO to COXs. Various concentrations of INDO and DM-INDO were addedto apo-proteins and the rate of fluorescence decrease was monitored overtime. After the mixture reached equilibrium, 50 μM of AA was added ascompetitor to monitor the reversibility of binding. The kinetic data forquenching were analyzed in the same manner as those for inhibition. Forclarity, the equilibrium constant for the first step of fluorescencequenching is referred to as K_(d), and k₂ and k⁻² from the second stepof quenching are represented by k′₂ and k′⁻², respectively.

As expected, INDO bound in a time-dependent fashion and was functionallyirreversible for all enzymes except V349L; the latter displayedreversible, time-dependent binding (FIG. 4). In agreement with thekinetics of inhibition, INDO quenched the V349A mutant more quickly thanwild type mCOX-2. INDO bound similarly to wild type mCOX-2 and V349I.Addition of AA could only compete INDO off V349L (FIG. 5D). The kineticparameters derived from INDO quenching correspond to those measured forINDO inhibition (Tables 6, 7).

DM-INDO displayed reversible, time-dependent binding with all fourenzymes in the quenching assay. The V349A mutation increased the rate ofbinding of DM-INDO, and also increased the apparent affinity (K_(d);Table 8). Strikingly, the value k′⁻² for V349A was the highest observedfor DM-INDO, almost sevenfold higher than the k′₂ of wild type mCOX-2(Table 8). The magnitude of this rate constant helps to explain whylittle or no inhibition was observed despite the fact that DM-INDO bindsto this enzyme. DM-INDO displayed a slightly smaller off-rate constant(k′⁻²) for V349I than wild type mCOX-2, which corresponded to the slightdifference in the activity recovery assay observed between these twoenzymes (Table 8, FIG. 3B). As with INDO, DM-INDO bound reversibly tothe V349L enzyme (FIG. 5). For each enzyme and inhibitor examined, thevalues of k′⁻² measured by fluorescence decay and calculated from theequation (2), were equal to the k′⁻² values measured by fluorescencerecovery upon incubation with AA (Tables 7 and 8). Overall, thefluorescence quenching kinetics agreed well with the kinetics ofinhibition.

TABLE 7 Kinetic Parameters of Fluorescence Quenching By INDO^(a) K_(d)k′₂ k′⁻² k′⁻² ^(b) Enzyme (μM) (s⁻¹) (s⁻¹) (s⁻¹) V349A  1.2 ± 0.3 0.12 ±0.01   ND^(c) ND wild type 12 ± 3 0.062 ± 0.009 ND ND mCOX-2 V349I 12 ±2 0.078 ± 0.008 ND ND V349L 15 ± 6 0.065 ± 0.012 0.0016 ± 0.0002 0.0020± 0.0001 ^(a)Kinetic parameters ± S.E. were determined from fluorescencequenching assays ^(b)Rate constant measured by fluorescence increasefrom competition with 50 μM AA. ^(c)Values were not detectable (ND).

TABLE 8 Kinetic Parameters of Fluorescence Quenching by DM-INDO^(a)K_(d) k′₂ k′⁻² k′⁻² ^(b) Enzyme (μM) (s⁻¹) (s⁻¹) (s⁻¹) V349A 1.4 ± 0.30.17 ± 0.04 0.045 ± 0.008 0.046 ± 0.002 wild type 16 ± 9  0.16 ± 0.040.006 ± 0.002 0.007 ± 0.001 mCOX-2 V349I 12 ± 5  0.09 ± 0.01 0.003 ±0.002 0.005 ± 0.001 V349L 34 ± 19 0.05 ± 0.01 0.003 ± 0.002 0.006 ±0.001 ^(a)Kinetic parameters ± S.E. were determined from fluorescencequenching assays. ^(b)Rate constants measured by fluorescence increasefrom competition with 50 μM AA.

A hallmark of INDO inhibition of COX enzymes is that it appears to befunctionally irreversible. Removal of the 2′ methyl group from INDOsignificantly reduces its potency as an inhibitor of COX-2 and COX-1 andeliminates its ability to serve as a functionally irreversibleinhibitor. In fact, ovine COX-1 does not appear to be inhibited at allby DM-INDO at concentrations up to 16 μM. Although the 2′ methyl groupappears to be a key contributor to time-dependent COX inhibition, it isnot the sole determinant of binding. Previous studies have demonstratedthat the carboxyl group of the indole-3-acetic acid moiety and thehalogen atom of the p-chlorobenzoyl group also contribute to COXinhibition by INDO. See Rome & Lands, 1975. Esterification or amidationof the carboxyl group transforms the molecule into a weak, reversibleinhibitor of COX-1 but does not eliminate time-dependent inhibition ofCOX-2 (Rome & Lands, 1975; Kalgutkar et al., 2000a). However, the modeof binding of INDO esters and amides appears to be different from thatof the parent acid. Inhibition of COX-2 by INDO is eliminated bymutations of Arg-120 or Tyr-355 whereas inhibition by INDO esters andamides is eliminated by mutations of Tyr-355 or Glu-524 but not Arg-120(Kalgutkar et al., 2000a). The absence of a strong ionic interaction canaccount for the slow reversibility of inhibition observed for certainINDO amides containing bulky substituents in the amide functional group,and for the inability of 2-des-methyl derivatives of INDO esters andamides to exhibit any inhibition of COX-2 (Kalgutkar et al., 2000b;Timofeevski et al., 2002).

The presently disclosed subject matter demonstrates the importance ofVal-349 in the interaction of COX enzymes with substrates andinhibitors. Previous studies have revealed that mutations of Val-349affect the affinities of COX-1 or COX-2 for substrates, the rates ofsubstrate oxygenation, and the regiochemistry and stereochemistry ofproduct formation (Thuresson et al., 2001; Schneider et al, 2002). Thedecreases in specific activity observed with the various mutants in thepresently disclosed subject matter are consistent with previous reports.The reduced activities of the mutants necessitated the adjustment ofprotein concentration in the inhibition assays to amounts that yieldedsimilar rates of substrate turnover. However, a constant proteinconcentration was used in the fluorescence quenching assays becausedifferences in specific activities were irrelevant due to the use ofapo-enzyme. Despite the differences in protein concentrations and otheraspects in the design of the inhibition and fluorescence quenchingassays, the values determined for the kinetic parameters of INDO andDM-INDO binding and inhibition were remarkably consistent with theexception of the rate-constants for dissociation where differences ofless than sevenfold were observed.

Pharmacological activities of INDO such as its anti-inflammatoryactivity, analgesic activity, and gastrointestinal toxicity are believedto derive from the ability of INDO to inhibit COX-2 and COX-1. However,INDO has been reported to exert additional biochemical activities incellular systems and it has been proposed that these non-COX activitiesmight contribute to its in vivo pharmacology (Weggen et al., 2001). Thepresently disclosed observation that a subtle chemicalmodification—modifying the 2′ methyl group to a moiety selected from thegroup consisting of hydrogen, halo, and C₂ to C₆ alkyl or branchedalkyl—greatly reduces COX inhibitory activity provides a strategy foroptimizing pharmacological effects at COX-independent targets whileminimizing undesirable side effects due to COX inhibition.

Example 12 Inhibition of COX Enzymes by 2-Des-Methyl Derivatives

The 2-Des-methyl analog of INDO (DM-INDO) was synthesized as discussedin Example 4, and tested against wild-type COX-1 and COX-2 as well asthe Val-349 mutants as described in Example 7. DM-INDO bound to allenzymes tested, but only displayed inhibitory potency against wild typemCOX-2 and the V349I enzyme. Without the 2′ methyl group anchoringDM-INDO in the active site, the compound was readily competed off of theenzyme by arachidonic acid (AA). The kinetics of inhibition werecomparable to the kinetics of binding as evaluated by fluorescencequenching. These results implicate the importance of the contactsbetween the 2′ methyl group of INDO and the “methyl-binding pocket”, inits time-dependent binding and inhibition of COXs.

Example 13 Cell Viability Assay

RKO and HCT-116 cells (human colorectal cancer cell lines) were culturedin microtiter plates (tissue culture grade, 96-well flat) in a finalvolume of 100 μl culture medium containing 5-8×10⁴ cells and the finalconcentrations of chemicals (1-500 μM). Cells were incubated in ahumidified atmosphere for 8-24 hours. To the cultures, 10 μl of cellproliferation reagent, WST-1 (Roche Applied Science, Indianapolis, Ind.,United States of America) was added, and reincubated for 1-2 hours. Theabsorbance of the samples was determined using a microtiter plate readerat a wavelength of 405-450 nm against background control. Referencewavelength was 620 nm. The tetrazolium salt, WST-1(4-[3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzenedisulfonate) was metabolized to formazan by the “succinate-tetrazoliumreductase” system, which exists in mitochondria and is active only inviable cells. Thus, the formation of formazan (dark yellow color) isproportional to the viable cell numbers. The results of this experimentare presented in FIG. 8.

Example 14 Determination of ED₅₀ Values for Derivatives

The cell viability assay described in Example 13 was employed with eachderivative listed in Table 9. The generated data points were used tocalculate ED₅₀ values by creating a sigmoidal dose-response curve usingnon-linear regression. ED₅₀ valued were calculated using the statisticalanalysis program PRISM® (GraphPad Software, Inc., San Diego, Calif.,United States of America).

The ED₅₀ values for the derivatives are presented in Table 9. ED₅₀values were also calculated for indomethacin and sulindac sulfide usingthe above referenced cell viability assay. Sulindac sulfide had an ED₅₀value of 98.2±1.4 μM in RKO cells, and 109.4±10.0 μM in HCT-116 cells.Indomethacin had an ED₅₀ value of 162.2±11.0 μM in RKO cells, and448.7±97.6 μM in HCT-116 cells.

TABLE 9 Activities of 2-Des-methyl Derivatives in Cell Viability Assays

ED₅₀ (μM) Compound R¹¹ R¹² RKO HCT-116 2

>3 >3 3

1.1 ± 0.7 3.9 ± 0.9 4

>3 >3 5

>3 >3 6

>3 >3 7

>3 >3 8

>3 >3 9

>3 >3 10

>3 >3 11

>3 >3 12

2.30 ± 0.03 >3 13

0.99 ± 0.03 2.08 ± 0.08 14

>3 >3 15

0.10 ± 0.04 1.2 ± 0.2 16

0.83 ± 0.06 1.47 ± 0.03 17

0.73 ± 0.06 1.2 ± 0.1 18

>3 >3 19

>3 >3 20

0.63 ± 0.07 0.8 ± 0.1 21

>3 >3 22

0.67 ± 0.07 1.3 ± 0.2 23

>3 >3 24

>3 >3 25

>3 >3 26

0.54 ± 0.07 2.1 ± 0.7 27

>3 >3 28

>3 >3 29

>3 >3 30

0.04 ± 0.02 0.18 ± 0.04 31

>3 >3

ED₅₀ (μm) Compound R¹² RKO HCT-116 32

2.4 ± 0.2 6 ± 1 33

0.8 ± 0.1 1.2 ± 0.3 34

5.4 ± 0.5 n.d. 35

0.7 ± 0.1 1.0 ± 0.8 36

6.4 ± 0.6 n.d. 37

>10 n.d. 38

6.7 ± 0.1 n.d. n.d.: not determined

Example 15 Caspase-3 Colorimetric Assay

RKO, HCT-116 (another human colorectal cancer cell line), and H1299 (ahuman non-small cell cancer line) cells that had been treated with testcompounds for various times were washed twice with ice-cold PBS andlysed in a lysis buffer (BioVision) on ice for 10 minutes, followed bycentrifugation at 15,000×g for 10 minutes. Caspase-3 activity, which isa measure if the initiation of apoptotic cell death, was determined inthe supernatant by a colorimeric assay kit (BioVision Inc., Palo Alto,Calif., United States of America) using the p-nitroanilide-labeledpeptide, DEVD-pNA, as substrate. Caspase-3 activity was monitored by therelease of p-nitroanilide from the substrate at 405 nm. The foldincrease in caspase-3 activity is shown in FIG. 9, and was calculated bycomparing the absorbance of p-nitroanilide from vehicle treated controlsand those treated with sulindac sulfide and its analogs.

Example 16 Preparation of Protein Lysates and Western Blotting Analysis

Cells that had been treated with test compounds for various times werewashed twice with ice-cold phosphate buffered saline (PBS) and lysed inkinase lysis buffer [50 mM Tris buffer (pH 7.5) 150 mM NaCl, 0.1% TritonX-100, 0.1% Nonidet P-40, 4 mM ethylenediamine tetraacetic acid (EDTA),50 mM NaF, 0.1 mM sodium orthovanadate, 1 mM dithiothreitol (DTT) andprotease inhibitors: antipain, leupeptin, pepstatin A, and chymostatin(5 μg/mL), phenylmethylsulfonyl fluoride (50 μg/mL) and4-(2-aminoethyl)-benzenesulfonylfluoride (100 μg/mL)] for 30 minutes at4° C. Cell lysates were cleared by centrifugation at 15,000 g for 15minutes, and the resulting supernatant was collected. Cellular protein(30-50 μg) was mixed with an equal volume of 2× Laemmli sample buffer[125 mM Tris (pH 6.8), 10% β-mercaptoethanol, 20% glycerol, 4% sodiumdodecyl sulfate (SDS), and 0.05% bromophenol blue] and boiled for 5minutes. The proteins were resolved by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) andelectrophoretically transferred onto polyvinylidene difluoride membranes(Millipore Corp., Bedford, Mass., United States of America). Themembranes were blocked with 5% non-fat milk in Tris-buffered saline (50mM Tris pH 7.5, 150 mM NaCl) containing 0.1% Tween 20, then incubatedwith anti-poly(ADP-ribose) polymerase (PharMingen, San Diego, Calif.,United States of America) for 1-2 hours. The primary antibody was thenstained with either donkey anti-rabbit or goat anti-mouse horseradishperoxidase-conjugate secondary antibodies. Enhanced chemiluminescencewas performed (ECL Western blotting detection system: AmershamBiosciences, Piscataway, N.J., United States of America) and proteinbands detected by autoradiography. The detection of a band correspondingto cleaved poly(ADP-ribose) polymerase indicated the initiation ofapoptotic cell death.

Example 17 PPARγ Reporter Assays

Human embryonic kidney cells (HEK293 cells) were purchased from AmericanType Culture Collection (ATCC) and maintained in Dulbecco's modifiedEagle's medium (DMEM) with GIBCO™ GLUTAMAX™ (Invitrogen Corp., Carlsbad,Calif., United States of America) and 10% heat-inactivated FBS (AtlasBiological, Fort Collins, Colo., United States of America) or 10%charcoal-stripped FBS (HyClone, Logan, Utah, United States of America)in a 5% CO₂ constant humidity 37° C. incubator. 9×10⁵ HEK293 cells wereplated in DMEM supplemented with 10% charcoal-stripped FBS. 18-24 hoursafter plating, cells were transfected with a control Renilla luciferaseexpressing plasmid (0.2 μg pCMV-renilla luciferase from Promega Corp.,Madison, Wis., United States of America), 0.4 μg PPARγ-GAL4, and 0.4 μgUAS-tk-luc using a lipid ratio of 8 μL Effectene Transfection Reagent(Qiagen Inc., Valencia, Calif., United States of America) per 1 μg DNAin 3 mL of DMEM+10% charcoal-stripped FBS. Cells remained in thetransfection mixture overnight until the transfection media was replacedwith DMEM+10% charcoal-stripped serum containing ligands of interest orvehicle (0.1% DMSO)

Transfected HEK293 cells were treated with various concentrations ofeindenic acid sulfide or the N-benzyl amide derivative of eindenic acidsulfide,N-Benzyl-2-[6-fluoro-3-(4-methylsulfanyl-benzylidene)-3H-inden-1-yl]-acetamide,dissolved in DMSO or vehicle alone (0.1% DMSO) for 4 hours. Cells werelysed in Passive Lysis Buffer (Promega Corp.) and lysates were assayedfor firefly luciferase and Renilla luciferase activity using theDUAL-LUCIFERASE® Reporter (DLR™) Assay system (Promega Corp., Catalog#E1910) according to the manufacturer's instructions. The results ofthis experiment are presented in FIG. 10.

Discussion of Example 17

2-Des-methylindomethacin and eindenic acid sulfide and a series ofstructural analogs were tested for their ability to induce apoptosis ofcultured cancer cells and for their ability to activate PPARγ-mediatedtranscription in transfected cells in culture. Both compounds weredemonstrated to be as active or more active than the parent drug. Infact, eindenic acid sulfide is considerably more active than sulindacsulfide in both assays. Similar results were obtained with analogs ofeindenic acid sulfide. While the co-inventors do not wish to be limitedto any particular theory of operation, the enhanced biological activityof 2-Des-methyl analogs might be due to the alteration in conformationthat results from deletion of the 2′ methyl group.

Example 18 Toxicity of 2-Des-methylindomethacin In Vivo

The toxicity of indomethacin and 2-Des-methylindomethacin were comparedin C57/BL6 mice, which are very sensitive to gastrointestinal toxicityby indomethacin. Daily injections of a series of concentrations ofindomethacin and 2-Des-methylindomethacin demonstrated that2-Des-methylindomethacin is at least 25-fold less toxic thanindomethacin, verifying that much higher concentrations of 2-Des-methylanalogs can be administered. Thus, 2-Des-methyl analogs of indomethacinand sulindac sulfide, as well as any prodrug forms (e.g., eindenic acidsulfoxide), appear to be attractive candidates for drugs targeting arange of diseases.

REFERENCES

The references listed below as well as all references cited in thespecification are incorporated herein by reference to the extent thatthey supplement, explain, provide a background for, or teachmethodology, techniques, and/or compositions employed herein.

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It will be understood that various details of the described subjectmatter can be changed without departing from the scope of the describedsubject matter. Furthermore, the foregoing description is for thepurpose of illustration only, and not for the purpose of limitation.

1. A method for inhibiting growth of a cell, the method comprisingcontacting the cell with a derivative of a compound, wherein thecompound comprises a cyclooxygenase inhibitor comprising an indoleaceticacid or indenacetic acid functional group having a 2′ methyl group andthe derivative substantially lacks cyclooxygenase inhibitory activity asa result of modifying the 2′ methyl group to a moiety selected from thegroup consisting of hydrogen; halo; halomethyl, wherein at least onehydrogen of the methyl group is substituted with a halogen; C₂ to C₆alkyl; C₂ to C₆ branched alkyl; and C₂ to C₆ substituted alkyl.
 2. Themethod of claim 1, wherein the cyclooxygenase inhibitor comprises anindenacetic acid functional group and the moiety is selected from thegroup consisting of hydrogen and fluorine.
 3. The method of claim 1,wherein the cell is present in a subject.
 4. The method of claim 3,wherein the cell is a tumor cell.
 5. The method of claim 4, wherein thesubject is a mammal.
 6. The method of claim 5, wherein the mammal is ahuman.
 7. The method of claim 1, wherein the compound is a non-steroidalanti-inflammatory drug.
 8. The method of claim 6, wherein thenon-steroidal anti-inflammatory drug is selected from the groupconsisting of indomethacin, sulindac, and pharmaceutically acceptablesalts thereof.
 9. A method for treating a disease in a subject, whereinthe disease is selected from the group consisting of a cancer, aneurodegenerative disease, and diabetes, the method comprisingadministering to the subject a treatment effective amount of aderivative of a compound, wherein the compound comprises acyclooxygenase inhibitor comprising an indoleacetic acid or indenaceticacid functional group having a 2′ methyl group and the derivativesubstantially lacks cyclooxygenase inhibitory activity as a result ofmodifying the 2′ methyl group to a moiety selected from the groupconsisting of hydrogen; halo; halomethyl, wherein at least one hydrogenof the methyl group is substituted with a halogen; C₂ to C₆ alkyl; C₂ toC₆ branched alkyl; and C₂ to C₆ substituted alkyl.
 10. The method ofclaim 9, wherein the cyclooxygenase inhibitor comprises an indenaceticacid functional group and the moiety is selected from the groupconsisting of hydrogen and fluorine.
 11. The method of claim 9, whereinthe subject is a mammal.
 12. The method of claim 11, wherein the mammalis a human.
 13. The method of claim 9, wherein the neurodegenerativedisease is Alzheimer's disease.
 14. The method of claim 9, wherein thecompound is a non-steroidal anti-inflammatory drug.
 15. The method ofclaim 14, wherein the non-steroidal anti-inflammatory drug is selectedfrom the group consisting of indomethacin and sulindac, pharmaceuticallyacceptable salts thereof, and combinations thereof.
 16. The method ofclaim 9, wherein the derivative causes substantially lessgastrointestinal toxicity than does the compound.
 17. A method forsuppressing tumor growth in a subject, the method comprisingadministering to a subject bearing a tumor a derivative of a compound,wherein the compound comprises a cyclooxygenase inhibitor comprising anindoleacetic acid or indenacetic acid functional group having a 2′methyl group and the derivative substantially lacks cyclooxygenaseinhibitory activity as a result of modifying the 2′ methyl group to amoiety selected from the group consisting of hydrogen; halo; halomethyl,wherein at least one hydrogen of the methyl group is substituted with ahalogen; C₂ to C₆ alkyl; C₂ to C₆ branched alkyl; and C₂ to C₆substituted alkyl.
 18. The method of claim 17, wherein thecyclooxygenase inhibitor comprises an indenacetic acid functional groupand the moiety is selected from the group consisting of hydrogen andfluorine.
 19. The method of claim 17, wherein the subject is a mammal.20. The method of claim 19, wherein the mammal is a human.
 21. Themethod of claim 17, wherein the compound is a non-steroidalanti-inflammatory drug.
 22. The method of claim 17, wherein thenon-steroidal anti-inflammatory drug is selected from the groupconsisting of indomethacin and sulindac, pharmaceutically acceptablesalts thereof, and combinations thereof.
 23. The method of claim 17,wherein the derivative causes substantially less gastrointestinaltoxicity than does the compound.
 24. A method for inducing apoptosis ina cell, the method comprising contacting the cell with a derivative of acompound, wherein the compound comprises a cyclooxygenase inhibitorcomprising an indoleacetic acid or indenacetic acid functional grouphaving a 2′ methyl group and the derivative substantially lackscyclooxygenase inhibitory activity as a result of modifying the 2′methyl group to a moiety selected from the group consisting of hydrogen;halo; halomethyl, wherein at least one hydrogen of the methyl group issubstituted with a halogen; C₂ to C₆ alkyl; C₂ to C₆ branched alkyl; andC₂ to C₆ substituted alkyl.
 25. The method of claim 24, wherein thecyclooxygenase inhibitor comprises an indenacetic acid functional groupand the moiety is selected from the group consisting of hydrogen andfluorine.
 26. The method of claim 24, wherein the cell is a cell inculture.
 27. The method of claim 24, wherein the cell is a cancer cell.28. The method of claim 24, wherein the cell is present within asubject.
 29. The method of claim 28, wherein the subject is a mammal.30. The method of claim 29, wherein the mammal is a human.
 31. Themethod of claim 24, wherein the compound is a non-steroidalanti-inflammatory drug.
 32. The method of claim 31, wherein thenon-steroidal anti-inflammatory drug is selected from the groupconsisting of indomethacin and sulindac, pharmaceutically acceptablesalts thereof, and combinations thereof.
 33. A method for modulating theactivity of a peroxisome proliferators activated receptor (PPAR)isoform, the method comprising contacting the PPAR isoform with aderivative of a compound, wherein the compound comprises acyclooxygenase inhibitor comprising an indoleacetic acid or indenaceticacid functional group having a 2′ methyl group and the derivativesubstantially lacks cyclooxygenase inhibitory activity as a result ofmodifying the 2′ methyl group to a moiety selected from the groupconsisting of hydrogen; halo; halomethyl, wherein at least one hydrogenof the methyl group is substituted with a halogen; C₂ to C₆ alkyl; C₂ toC₆ branched alkyl; and C₂ to C₆ substituted alkyl.
 34. The method ofclaim 33, wherein the cyclooxygenase inhibitor comprises an indenaceticacid functional group and the moiety is selected from the groupconsisting of hydrogen and fluorine.
 35. The method of claim 33, whereinthe peroxisome proliferators activated receptor (PPAR) isoform is PPARγ.36. The method of claim 33, wherein the peroxisome proliferatorsactivated receptor (PPAR) isoform is present in a subject.
 37. Themethod of claim 36, wherein the subject is a mammal.
 38. The method ofclaim 37, wherein the mammal is a human.
 39. The method of claim 33,wherein the compound is a non-steroidal anti-inflammatory drug.
 40. Themethod of claim 33, wherein the non-steroidal anti-inflammatory drug isselected from the group consisting of indomethacin and sulindac,pharmaceutically acceptable salts thereof, and combinations thereof. 41.The method of claim 33, wherein the derivative causes substantially lessgastrointestinal toxicity than does the compound.
 42. A method foraltering specificity of a cyclooxygenase-inhibiting compound, the methodcomprising: (a) providing a compound having cyclooxygenase inhibitoryactivity, the compound comprising an indoleacetic acid or indenaceticacid functional group having a 2′ methyl group; and (b) replacing the 2′methyl group with a moiety selected from the group consisting ofhydrogen; halo; halomethyl, wherein at least one hydrogen of the methylgroup is substituted with a halogen; C₂ to C₆ alkyl; C₂ to C₆ branchedalkyl; and C₂ to C₆ substituted alkyl to create a derivative, whereinthe derivative substantially lacks cyclooxygenase inhibitory activity.43. The method of claim 42, wherein the compound comprises anindenacetic acid functional group and the moiety is selected from thegroup consisting of hydrogen and fluorine.
 44. The method of claim 42,wherein the compound is a non-steroidal anti-inflammatory drug.
 45. Themethod of claim 44, wherein the non-steroidal anti-inflammatory drug isselected from the group consisting of indomethacin and sulindac,pharmaceutically acceptable salts thereof, and combinations thereof. 46.The method of any of claims 1, 9, 17, 24, and 33, wherein the derivativehas one of the following formulas:

wherein R² is selected from the group consisting of hydrogen, halo, CF₃;SCH₃; SOCH₃; SO₂CH₃; SO₂NH₂; CONH₂; C₁ to C₆ alkyl, branched alkyl, orsubstituted alkyl; C₁ to C₆ alkoxy, branched alkoxy, or substitutedalkoxy; benzyloxy; C₁ to C₆ alkylcarboxylic acid, branchedalkylcarboxylic acid, or substituted alkylcarboxylic acid; and CH₂N₃; R⁴is selected from the group consisting of hydrogen; halo; CF₃; C₁ to C₆alkyl, branched alkyl, or substituted alkyl; C₁ to C₆ alkoxy, branchedalkoxy, or substituted alkoxy; aryl; substituted aryl; benzyloxy; SCH₃;SOCH₃; SO₂CH₃; and SO₂NH₂; R⁵ is selected from the group consisting ofhydrogen, C₁ to C₆ alkyl, C₁ to C₆ branched alkyl, and C₁ to C₆substituted alkyl, and in the case of Formula Ia, ═O; R⁶ is selectedfrom the group consisting of hydrogen; C₁ to C₆ alkyl, branched alkyl,or substituted alkyl; C₁ to C₆ alkoxy, branched alkoxy, or substitutedalkoxy; benzyloxy; C₁ to C₆ alkylcarboxylic acid, branchedalkylcarboxylic acid, or substituted alkylcarboxylic acid; and thefollowing structure:

wherein Ar is cyclohexyl or phenyl; R⁷ is hydrogen; C₁ to C₆ alkyl,branched alkyl, or substituted alkyl; R⁸ is hydrogen, halo, C₁ to C₆alkyl, branched alkyl, and substituted alkyl; C₁ to C₆ alkoxy, branchedalkoxy, and substituted alkoxy; C₁ to C₆ alkylcarboxylic acid, branchedalkylcarboxylic acid, or substituted alkylcarboxylic acid; amino; nitro;CF₃; bromoacetamidyl; benzoyl; or 2-phenyl-oxiranyl; X is O or NR⁹,wherein R⁹ is hydrogen or alkyl; and m, n, and t are each individually0, 1, 2, 3, 4, or 5; R¹⁵ is selected from the group consisting ofcyclohexyl and an aromatic substituent, wherein the aromatic substituentcomprises an aryl, a heteroaryl, and singly or multiply substitutedderivatives thereof; Y is selected from the group consisting ofhydrogen; halo; halomethyl, wherein at least one hydrogen of the methylgroup is substituted with a halogen; C₂ to C₆ alkyl; C₂ to C₆ branchedalkyl; and C₂ to C₆ substituted alkyl; and q is 0, 1, 2, 3, or
 4. 47.The method of claim 46, wherein R¹⁵ comprises an aryl comprisingmultiple aromatic rings that are fused together or covalently linked.48. The method of claim 47, the multiple aromatic rings are covalentlylinked by a moiety selected from the group consisting of an alkylenemoiety, a carbonyl, oxygen, diphenylether, and nitrogen.
 49. The methodof claim 47, wherein the multiple aromatic rings are selected from thegroup consisting of naphthyl, biphenyl, diphenylether, diphenylamine,and benzophenone.
 50. The method of claim 46, wherein the derivative isselected from the group consisting of 2-Des-methylindomethacin, eindenicacid sulfide, eindenic acid sulfoxide, and eindenic acid sulfone. 51.The method of claim 46, wherein the derivative is eindenic acid sulfide.52. The method of claim 46, further comprising derivatizing a carboxylicacid moiety present on the compound to an ester or an amide.
 53. Themethod of claim 52, wherein the ester or amide has a structure presentedin Table
 3. 54. A compound of the following formula:

wherein R² is selected from the group consisting of hydrogen, halo, CF₃;SCH₃; SOCH₃; SO₂CH₃; SO₂NH₂; CONH₂; C₁ to C₆ alkyl, branched alkyl, orsubstituted alkyl; C₁ to C₆ alkoxy, branched alkoxy, or substitutedalkoxy; benzyloxy; C₁ to C₆ alkylcarboxylic acid, branchedalkylcarboxylic acid, or substituted alkylcarboxylic acid; and CH₂N₃; R⁴is selected from the group consisting of hydrogen; halo; CF₃; C₁ to C₆alkyl, branched alkyl, or substituted alkyl; C₁ to C₆ alkoxy, branchedalkoxy, or substituted alkoxy; aryl; substituted aryl; benzyloxy; SCH₃;SOCH₃; SO₂CH₃; and SO₂NH₂; R⁵ is selected from the group consisting ofhydrogen, C₁ to C₆ alkyl, C₁ to C₆ branched alkyl, and C₁ to C₆substituted alkyl; R⁶ is selected from the group consisting of C₁ to C₆alkylcarboxylic acid, branched alkylcarboxylic acid, and substitutedalkylcarboxylic acid R¹⁵ is selected from the group consisting ofcyclohexyl and an aromatic substituent, wherein the aromatic substituentcomprises an aryl, a heteroaryl, and singly or multiply substitutedderivatives thereof; Y is selected from the group consisting ofhydrogen; halo; halomethyl, wherein at least one hydrogen of the methylgroup is substituted with a halogen; C₂ to C₆ alkyl; C₂ to C₆ branchedalkyl; and C₂ to C₆ substituted alkyl; and q is 0, 1, 2, 3, or
 4. 55.The compound of claim 54, wherein R¹⁵ comprises an aryl comprisingmultiple aromatic rings that are fused together or covalently linked.56. The compound of claim 55, the multiple aromatic rings are covalentlylinked by a moiety selected from the group consisting of an alkylenemoiety, a carbonyl, oxygen, diphenylether, and nitrogen.
 57. Thecompound of claim 55, wherein the multiple aromatic rings are selectedfrom the group consisting of naphthyl, biphenyl, diphenylether,diphenylamine, and benzophenone.
 58. The compound of claim 54, whereinR² is selected from the group consisting of halo, C₁ to C₆ alkyl orbranched alkyl, SCH₃, SOCH₃, SO₂CH₃, and SO₂NH₂.
 59. The compound ofclaim 58, wherein R² is F.
 60. The compound of claim 54, wherein R¹⁵ isin the E orientation with respect to the indene ring.
 61. The compoundof claim 54, wherein the R¹⁵ comprises one of the following formulas:


62. The compound of claim 54, wherein the compound has one of thefollowing formulas: